Pharmaceutically acceptable salts of fatty acids

ABSTRACT

The present disclosure provides pharmaceutically acceptable stable salt forms of 15-lipoxygenase products, such as 15-HETrE lysine salt, compositions comprising same and methods of making and using same.

PRIORITY CLAIM

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/904,600, filed Nov. 15, 2013, the entire contents of whichare incorporated herein by reference and relied upon.

FIELD

The disclosure generally relates to pharmaceutically acceptable stablesalt forms of 15-lipoxygenase products.

BACKGROUND

Enzymes are highly selective catalysts, greatly accelerating both therate and specificity of biochemical reactions. In enzymatic reactions,the molecules at the beginning of the process, called substrates, areconverted into different molecules, called products.

Lipoxygenases are enzymes that catalyze the oxidation of polyunsaturatedfatty acids. 15-lipoxygenase (15-LOX) is one such enzyme that catalyzesthe oxidation of substrates such as linolenic acid, dihomo-gammalinolenic acid, eicosapentaenoic acid and arachidonic acid to therespective products 13-HODE, 15-HETrE, 15-OHEPA and 15-HETE.

15-LOX is responsible for the conversion of arachidonic acid to variousbiologically active metabolites including15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE). 15-HETE has beenimplicated in the pathogenesis of airway and allergic diseases such asasthma by contributing to bronchoconstriction, mucus secretion, andeosinophil migration. While 15-HETE has been implicated inproinflammatory reactions, other 15-LOX products, such as 15-OHEPA,15-HETrE and 13-HODE, have been shown to have anti-inflammatory effectsand may be medically useful.

Examples of potential medically useful indications for 15-LOX products,incorporated by reference, include, but are not limited to acne therapy(U.S. Pat. No. 8,293,790), erythema therapy (US patent applicationpublication 2013/0101533), anti-microbials (US patent applicationpublication 2012/0264705), fatty liver therapy (GB1300628.3), neuropathytherapy (WO2010/125330A1) and treatment for skin inflammation (U.S. Pat.No. 8,536,223).

The products of 15-LOX metabolism are unstable at room temperature andneed to be stored at temperatures of −20° C. or less. Due to thisinstability, 15-LOX products have not been formulated for medical use.As shown below, an issue with these compounds is the tendency for the15-LOX derived hydroxyl group to form fatty acid dimers by forming esterbonds with the carboxyl group of the fatty acid.

Because of the instability and dimer formation, their potential to bedeveloped as a medicine has been a challenge. Pharmaceuticallyacceptable stable forms of 15-LOX products are described herein.

SUMMARY

The present disclosure provides compositions comprising stable,pharmaceutically useful salts of 15-lipoxygenase fatty acid products.

The invention also provides pharmaceutical compositions comprising anyof the compounds described herein and a pharmaceutically acceptablecarrier.

In some embodiments the, the fatty acids are 15-HETrE, 15-OHEPA or13-HODE. In another embodiment the salt forms are sodium or lysine saltsof 15-HETrE, 15-OHEPA or 13-HODE. In a specific embodiment, theinvention is a lysine salt of 15-HETrE.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows the X-ray powder diffraction pattern of 15-HETrE.

FIG. 2 shows the differential scanning calorimetry (DSC) thermogram of15-HETrE from −70° C. to 200° C. at a rate of 10° C./min.

FIG. 3 shows the hyper-differential scanning calorimetry (Hyper DSC)thermogram of 15-HETrE from −75° C. to 150° C. at a rate of 200° C./min.

FIG. 4 shows the proton-NMR spectrum of HETrE in d₄-methanol at 500 MHz.

FIG. 5 shows the FT-IR spectrum of 15-HETrE.

FIG. 6 shows a comparison of the X-ray powder diffraction patterns of15-HETrE in free acid form (top panel) with sodium hydrogen carbonate(middle panel) and 15-HETrE sodium salt (bottom panel).

FIG. 7 shows a comparison of the proton NMR spectra of 15-HETrE in freeacid form (top spectrum) and 15-HETrE sodium salt (bottom spectrum).

FIG. 8 shows the FT-IR spectrum of 15-HETrE sodium salt.

FIG. 9 shows the differential scanning calorimetry (DSC) thermogram of15-HETrE sodium salt.

FIG. 10 shows a comparison of the X-ray powder diffraction patterns of15-HETrE free acid (top panel), lysine prepared by salt cracking (secondpanel down), lysine monohydrate (third panel down), and 15-HETrE lysinesalt (bottom panel).

FIG. 11 shows a comparison of the proton NMR spectra for lysine (topspectrum), 15-HETrE free acid (middle spectrum) and 15-HETrE lysine salt(bottom spectrum).

FIG. 12 shows the FT-IR spectrum of 15-HETrE lysine salt.

FIG. 13 shows the differential scanning calorimetry (DSC) thermogram of15-HETrE lysine salt.

FIG. 14 shows a comparison of the X-ray powder diffraction patterns of15-HETrE free acid (top panel), ornithine prepared by salt cracking(middle panel), and 15-HETrE ornithine salt (bottom panel).

FIG. 15 shows a comparison of the proton NMR spectra for ornithine (topspectrum), 15-HETrE free acid (middle spectrum) and 15-HETrE ornithinesalt (bottom spectrum).

FIG. 16 shows the FT-IR spectrum of 15-HETrE ornithine salt.

FIG. 17 shows the differential scanning calorimetry (DSC) thermogram of15-HETrE ornithine salt from 30° C. to 300° C. at a rate of 10° C./min.

FIG. 18 shows a comparison of the X-ray powder diffraction patterns of15-HETrE free acid (top panel), piperazine (middle panel), and 15-HETrEpiperazine salt (bottom panel).

FIG. 19 shows a comparison of the proton NMR spectra for 15-HETrE freeacid (top spectrum) and 15-HETrE piperazine salt (bottom spectrum).

FIGS. 20A-H show 10× magnified images of 15-HETrE lysine salt with firstorder red plate (FIG. 20A) and with crossed-polarized light (FIG. 20B).15-HETrE sodium salt with first order red plate (FIG. 20C) and withcrossed-polarized light (FIG. 20D). 15-HETrE ornithine salt with firstorder red plate (FIG. 20E) and with crossed-polarized light (FIG. 20F).and 15-HETrE piperazine salt with first order red plate (FIG. 20G) andwith crossed-polarized light (FIG. 20H).

FIG. 21 shows a comparison of X-ray powder diffraction patterns for15-HETrE lysine salt before (top panel) and after (bottom panel) dynamicvapor sorption (DVS) analysis.

FIG. 22 shows the proton NMR spectrum of 15-HETrE lysine salt ind₄-methanol.

FIG. 23 shows a thermogravimetric/differential thermal analysis(“TG/DTA”) thermogram of 15-HETrE lysine salt.

FIG. 24 shows the differential scanning calorimetry (DSC) thermogram of15-HETrE lysine salt from 30° C. to 300° C. at a rate of 10° C./min.

FIG. 25A shows hotstage microscopy images of 15-HETrE lysine salt at 30°C.

FIG. 25B shows hotstage microsopy images of 15-HETrE lysine salt at 120°C.

FIG. 25C shows hotstage microsopy images of 15-HETrE lysine salt at 200°C.

FIG. 25D shows hotstage microsopy images of 15-HETrE lysine salt atambient temperature after melt.

FIG. 26 shows the dynamic vapor sorption (DVS) isotherm of 15-HETrElysine salt.

FIG. 27 shows the FT-IR spectrum of 15-HETrE lysine salt.

FIG. 28 shows the stability of 15-HETrE free acid (“15-HETrE FFA”) whenstored for up to 24 weeks at −20° C., at 2-8° C., and at 25° C., and15-HETrE lysine salt when stored capped under nitrogen gas at 40° C. and75% RH (“15-HETrE Lysine Salt 40/75”) and at 20° C. and 60% RH(“15-HETrE Lysine Salt 20/60”).

FIG. 29 shows a comparison of X-ray powder diffraction patterns forsolids isolated from 15-HETrE and meglumine (top panel) and meglumine(bottom panel).

FIG. 30 shows the proton NMR spectrum of solids isolated from 15-HETrEand meglumine.

FIGS. 31A-B show 10× photomicrographs of 15-HETrE meglumine saltcaptured with first order red plate (FIG. 31A) and withcrossed-polarized light (FIG. 31B).

DETAILED DESCRIPTION

13-HODE is formed by the action of 15-LOX on linoleic acid. Linoleicacid is oxidized to 13-hydroperoxyoctadeca-9Z,11E-dienoic acid(13-HODE). As used herein, the term “13-HODE” refers to 13-HODE in itsfree acid form.

15-Hydroxy-eicosa-8(Z),11(Z),13(E)-trienoic acid (“15-HETrE” or “HETrE”)is a 15-LOX derivative of dihomo-gamma linolenic acid (DGLA). 15-HETrEcan be synthesized from DGLA according to methods in the art. As usedherein, the term “15-HETrE” refers to 15-HETrE in its free acid form(e.g, 15-hydroxy-eicosa-8(Z),11(Z),13(E)-trienoic acid).

15-Hydroxy-eicosa-5(Z),8(Z),11(Z),13(E),17(Z)-pentaenoic acid(“15-OHEPA”) is a 15-LOX derivative of EPA. 15-OHEPA can be synthesizedfrom EPA according to methods known in the art. As used herein, the term“15-OHEPA” refers to 15-OHEPA in its free acid form (e.g,15-hydroxy-eicosa-5,8,11,13,17-pentaenoic acid).

As used herein, the term “pharmaceutically useful” refers to compoundsand/or compositions that are chemically and physically compatible (e.g.,substantially miscible and/or nonreactive) with other pharmaceuticalcomponents, or to pharmaceutical compositions that are generally stableunder common storage conditions (e.g., room temperature or refrigerated)for a length of time sufficient to provide a commercially viable shelflife.

In various embodiments, the invention provides pharmaceuticalcompositions, for example topically deliverable compositions, comprisingone or more of 13-HODE, 15-OHEPA, 15-HETrE or mixtures thereof.

In one embodiment, the present disclosure provides topicalpharmaceutical compositions comprising, for example, an amount (e.g., atherapeutically effective amount) of a salt form of 13-HODE, 15-OHEPA,15-HETrE, or a combination thereof. In one embodiment, thepharmaceutical composition comprises about 0.1 wt. % to about 20 wt. %of the 13-HODE, 15-OHEPA, 15-HETrE, or a combination thereof, forexample about 0.1 wt. %, about 0.2 wt. %, about 0.3 wt. %, about 0.4 wt.%, about 0.5 wt. %, about 0.6 wt. %, about 0.7 wt. %, about 0.8 wt. %,about 0.9 wt. %, about 1 wt. %, about 1.1 wt. %, about 1.2 wt. %, about1.3 wt. %, about 1.4 wt. %, about 1.5 wt. %, about 1.6 wt. %, about 1.7wt. %, about 1.8 wt. %, about 1.9 wt. %, about 2 wt. %, about 2.1 wt. %,about 2.2 wt. %, about 2.3 wt. %, about 2.4 wt. %, about 2.5 wt. %,about 2.6 wt. %, about 2.7 wt. %, about 2.8 wt. %, about 2.9 wt. %,about 3 wt. %, about 3.1 wt. %, about 3.2 wt. %, about 3.3 wt. %, about3.4 wt. %, about 3.5 wt. %, about 3.6 wt. %, about 3.7 wt. %, about 3.8wt. %, about 3.9 wt. %, about 4 wt. %, about 4.1 wt. %, about 4.2 wt. %,about 4.3 wt. %, about 4.4 wt. %, about 4.5 wt. %, about 4.6 wt. %,about 4.7 wt. %, about 4.8 wt. %, about 4.9 wt. %, about 5 wt. %, about5.1 wt. %, about 5.2 wt. %, about 5.3 wt. %, about 5.4 wt. %, about 5.5wt. %, about 5.6 wt. %, about 5.7 wt. %, about 5.8 wt. %, about 5.9 wt.%, about 6 wt. %, about 6.1 wt. %, about 6.2 wt. %, about 6.3 wt. %,about 6.4 wt. %, about 6.5 wt. %, about 6.6 wt. %, about 6.7 wt. %,about 6.8 wt. %, about 6.9 wt. %, about 7 wt. %, about 7.1 wt. %, about7.2 wt. %, about 7.3 wt. %, about 7.4 wt. %, about 7.5 wt. %, about 7.6wt. %, about 7.7 wt. %, about 7.8 wt. %, about 7.9 wt. %, about 8 wt. %,about 8.1 wt. %, about 8.2 wt. %, about 8.3 wt. %, about 8.4 wt. %,about 8.5 wt. %, about 8.6 wt. %, about 8.7 wt. %, about 8.8 wt. %,about 8.9 wt. %, about 9 wt. %, about 9.1 wt. %, about 9.2 wt. %, about9.3 wt. %, about 9.4 wt. %, about 9.5 wt. %, about 9.6 wt. %, about 9.7wt. %, about 9.8 wt. %, about 9.9 wt. %, about 10 wt. %, about 10.1 wt.%, about 10.2 wt. %, about 10.3 wt. %, about 10.4 wt. %, about 10.5 wt.%, about 10.6 wt. %, about 10.7 wt. %, about 10.8 wt. %, about 10.9 wt.%, about 11 wt. %, about 11.1 wt. %, about 11.2 wt. %, about 11.3 wt. %,about 11.4 wt. %, about 11.5 wt. %, about 11.6 wt. %, about 11.7 wt. %,about 11.8 wt. %, about 11.9 wt. %, about 12 wt. %, about 12.1 wt. %,about 12.2 wt. %, about 12.3 wt. %, about 12.4 wt. %, about 12.5 wt. %,about 12.6 wt. %, about 12.7 wt. %, about 12.8 wt. %, about 12.9 wt. %,about 13 wt. %, about 13.1 wt. %, about 13.2 wt. %, about 13.3 wt. %,about 13.4 wt. %, about 13.5 wt. %, about 13.6 wt. %, about 13.7 wt. %,about 13.8 wt. %, about 13.9 wt. %, about 14 wt. %, about 14.1 wt. %,about 14.2 wt. %, about 14.3 wt. %, about 14.4 wt. %, about 14.5 wt. %,about 14.6 wt. %, about 14.7 wt. %, about 14.8 wt. %, about 14.9 wt. %,about 15 wt. %, about 15.1 wt. %, about 15.2 wt. %, about 15.3 wt. %,about 15.4 wt. %, about 15.5 wt. %, about 15.6 wt. %, about 15.7 wt. %,about 15.8 wt. %, about 15.9 wt. %, about 16 wt. %, about 16.1 wt. %,about 16.2 wt. %, about 16.3 wt. %, about 16.4 wt. %, about 16.5 wt. %,about 16.6 wt. %, about 16.7 wt. %, about 16.8 wt. %, about 16.9 wt. %,about 17 wt. %, about 17.1 wt. %, about 17.2 wt. %, about 17.3 wt. %,about 17.4 wt. %, about 17.5 wt. %, about 17.6 wt. %, about 17.7 wt. %,about 17.8 wt. %, about 17.9 wt. %, about 18 wt. %, about 18.1 wt. %,about 18.2 wt. %, about 18.3 wt. %, about 18.4 wt. %, about 18.5 wt. %,about 18.6 wt. %, about 18.7 wt. %, about 18.8 wt. %, about 18.9 wt. %,about 19 wt. %, about 19.1 wt. %, about 19.2 wt. %, about 19.3 wt. %,about 19.4 wt. %, about 19.5 wt. %, about 19.6 wt. %, about 19.7 wt. %,about 19.8 wt. %, about 19.9 wt. %, or about 20 wt. % of the 13-HODE,15-OHEPA, 15-HETrE, or a combination thereof.

Any pharmaceutically acceptable excipient known to those of skill in theart may be used in pharmaceutical compositions according to the presentdisclosure. Any excipient selected for use in the therapeutic andcosmetic compositions should be pharmaceutically and/or cosmeticallyacceptable and appropriate for the form in which the therapeuticcomposition will be used, e.g., cream, gel, milk, oil, lotion, and thelike. Preferably, the excipient has an affinity for the skin, is welltolerated, and stable when used in an amount adequate to provide thedesired consistency and ease of application. By way of example only, apharmaceutical composition according to the present disclosure maycomprise one or more of: surfactants, preservatives, flavoring agents,co-solvents, viscosity aids, suspension aids, and lipophilic phases.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a therapeutically effective amount of a salt formof 15-HETrE, 15-OHEPA or 13-HODE. The salt form of 15-HETrE, 15-OHEPA or13-HODE may be the sole significant active ingredient in thatcomposition and in the methods and uses as stated herein. The salt formof 15-HETrE, 15-OHEPA or 13-HODE may be the sole active ingredient.Alternatively, the salt form of 15-HETrE, 15-OHEPA or 13-HODE may becombined for co-formulation or co-administration with other agents fortreating a disease or disorder. If an additional active agent is to beused, the salt form of 15-HETrE, 15-OHEPA or 13-HODE can beco-formulated as a single dosage unit or can be formulated as two to aplurality of dosage units for coordinated, combination or concomitantadministration.

In various embodiments, the invention provides pharmaceuticalcompositions, for example orally deliverable compositions, comprisingthe salt form of 15-HETrE, 15-OHEPA or 13-HODE. In one embodiment, thecompositions comprise a therapeutically effective amount of the saltform of 15-HETrE, 15-OHEPA or 13-HODE. In one embodiment, thepharmaceutical composition comprises about 0.1% to about 99.9%, about 1%to about 95%, or about 5% to about 90% by weight of the salt form of15-HETrE, 15-OHEPA or 13-HODE, for example about 1%, about 2%, about 3%,about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%,about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%,about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%,about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%,about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%,about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%,about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%,about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about99.9% by weight of the salt form of 15-HETrE, 15-OHEPA or 13-HODE.

In one embodiment, the pharmaceutical composition comprises about atleast about 70%, at least about 71%, at least about 72%, at least about73%, at least about 74%, at least about 75%, at least about 76%, atleast about 77%, at least about 78%, at least about 79%, at least about80%, at least about 81%, at least about 82%, at least about 83%, atleast about 84%, at least about 85%, at least about 86%, at least about87%, at least about 88%, at least about 89%, at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, at least about 99.5%, or about99.9%, by weight, of the salt form of 15-HETrE, 15-OHEPA or 13-HODE.

In one embodiment, the pharmaceutical composition comprises at leastabout 50%, at least about 60%, at least about 70%, at least about 80% orat least about 90%, by weight of the salt form of 15-HETrE, 15-OHEPA or13-HODE.

In another embodiment, the salt form of 15-HETrE, 15-OHEPA or 13-HODE ispresent in a composition of the invention in an amount of about 1 mg toabout 10,000 mg, about 25 mg to about 7500 mg, about 25 mg to about 5000mg, about 50 mg to about 5000 mg, about 50 mg to about 3000 mg, about 75mg to about 2500 mg, or about 100 mg to about 1000 mg, for example about1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg,about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg,about 24 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about875 mg, about 900 mg, about 925 mg, about 950 mg, about 975 mg, about1000 mg, about 1025 mg, about 1050 mg, about 1075 mg, about 1100 mg,about 1025 mg, about 1050 mg, about 1075 mg, about 1200 mg, about 1225mg, about 1250 mg, about 1275 mg, about 1300 mg, about 1325 mg, about1350 mg, about 1375 mg, about 1400 mg, about 1425 mg, about 1450 mg,about 1475 mg, about 1500 mg, about 1525 mg, about 1550 mg, about 1575mg, about 1600 mg, about 1625 mg, about 1650 mg, about 1675 mg, about1700 mg, about 1725 mg, about 1750 mg, about 1775 mg, about 1800 mg,about 1825 mg, about 1850 mg, about 1875 mg, about 1900 mg, about 1925mg, about 1950 mg, about 1975 mg, about 2000 mg, about 2025 mg, about2050 mg, about 2075 mg, about 2100 mg, about 2125 mg, about 2150 mg,about 2175 mg, about 2200 mg, about 2225 mg, about 2250 mg, about 2275mg, about 2300 mg, about 2325 mg, about 2350 mg, about 2375 mg, about2400 mg, about 2425 mg, about 2450 mg, about 2475 mg, about 2500 mg,2525 mg, about 2550 mg, about 2575 mg, about 2600 mg, about 2625 mg,about 2650 mg, about 2675 mg, about 2700 mg, about 2725 mg, about 2750mg, about 2775 mg, about 2800 mg, about 2825 mg, about 2850 mg, about2875 mg, about 2900 mg, about 2925 mg, about 2950 mg, about 2975 mg,about 3000 mg, about 3025 mg, about 3050 mg, about 3075 mg, about 3100mg, about 3125 mg, about 3150 mg, about 3175 mg, about 3200 mg, about3225 mg, about 3250 mg, about 3275 mg, about 3300 mg, about 3325 mg,about 3350 mg, about 3375 mg, about 3400 mg, about 3425 mg, about 3450mg, about 3475 mg, about 3500 mg, about 3525 mg, about 3550 mg, about3575 mg, about 3600 mg, about 3625 mg, about 3650 mg, about 3675 mg,about 3700 mg, about 3725 mg, about 3750 mg, about 3775 mg, about 3800mg, about 3825 mg, about 3850 mg, about 3875 mg, about 3900 mg, about3925 mg, about 3950 mg, about 3975 mg, about 4000 mg, about 4025 mg,about 4050 mg, about 4075 mg, about 4100 mg, about 4125 mg, about 4150mg, about 4175 mg, about 4200 mg, about 4225 mg, about 4250 mg, about4275 mg, about 4300 mg, about 4325 mg, about 4350 mg, about 4375 mg,about 4400 mg, about 4425 mg, about 4450 mg, about 4475 mg, about 4500mg, about 4525 mg, about 4550 mg, about 4575 mg, about 4600 mg, about4625 mg, about 4650 mg, about 4675 mg, about 4700 mg, about 4725 mg,about 4750 mg, about 4775 mg, about 4800 mg, about 4825 mg, about 4850mg, about 4875 mg, about 4900 mg, about 4925 mg, about 4950 mg, about4975 mg, about 5000 mg, about 5025 mg, about 5050 mg, about 5075 mg,about 5100 mg, about 5125 mg, about 5150 mg, about 5175 mg, about 5200mg, about 5225 mg, about 5250 mg, about 5275 mg, about 5300 mg, about5325 mg, about 5350 mg, about 5375 mg, about 5400 mg, about 5425 mg,about 5450 mg, about 5475 mg, about 5500 mg, about 5525 mg, about 5550mg, about 5575 mg, about 5600 mg, about 5625 mg, about 5650 mg, about5675 mg, about 5700 mg, about 5725 mg, about 5750 mg, about 5775 mg,about 5800 mg, about 5825 mg, about 5850 mg, about 5875 mg, about 5900mg, about 5925 mg, about 5950 mg, about 5975 mg, about 6000 mg, about6025 mg, about 6050 mg, about 6075 mg, about 6100 mg, about 6125 mg,about 6150 mg, about 6175 mg, about 6200 mg, about 6225 mg, about 6250mg, about 6275 mg, about 6300 mg, about 6325 mg, about 6350 mg, about6375 mg, about 6400 mg, about 6425 mg, about 6450 mg, about 6475 mg,about 6500 mg, about 6525 mg, about 6550 mg, about 6575 mg, about 6600mg, about 6625 mg, about 6650 mg, about 6675 mg, about 6700 mg, about6725 mg, about 6750 mg, about 6775 mg, about 6800 mg, about 6825 mg,about 6850 mg, about 6875 mg, about 6900 mg, about 6925 mg, about 6950mg, about 6975 mg, about 7000 mg, about 7025 mg, about 7050 mg, about7075 mg, about 7100 mg, about 7125 mg, about 7150 mg, about 7175 mg,about 7200 mg, about 7225 mg, about 7250 mg, about 7275 mg, about 7300mg, about 7325 mg, about 7350 mg, about 7375 mg, about 7400 mg, about7425 mg, about 7450 mg, about 7475 mg, about 7500 mg, about 7525 mg,about 7550 mg, about 7575 mg, about 7600 mg, about 7625 mg, about 7650mg, about 7675 mg, about 7700 mg, about 7725 mg, about 7750 mg, about7775 mg, about 7800 mg, about 7825 mg, about 7850 mg, about 7875 mg,about 7900 mg, about 7925 mg, about 7950 mg, about 7975 mg, about 8000mg, about 8025 mg, about 8050 mg, about 8075 mg, about 8100 mg, about8125 mg, about 8150 mg, about 8175 mg, about 8200 mg, about 8225 mg,about 8250 mg, about 8275 mg, about 8300 mg, about 8325 mg, about 8350mg, about 8375 mg, about 8400 mg, about 8425 mg, about 8450 mg, about8475 mg, about 8500 mg, about 8525 mg, about 8550 mg, about 8575 mg,about 8600 mg, about 8625 mg, about 8650 mg, about 8675 mg, about 8700mg, about 8725 mg, about 8750 mg, about 8775 mg, about 8800 mg, about8825 mg, about 8850 mg, about 8875 mg, about 8900 mg, about 8925 mg,about 8950 mg, about 8975 mg, about 9000 mg, about 9025 mg, about 9050mg, about 9075 mg, about 9100 mg, about 9125 mg, about 9150 mg, about9175 mg, about 9200 mg, about 9225 mg, about 9250 mg, about 9275 mg,about 9300 mg, about 9325 mg, about 9350 mg, about 9375 mg, about 9400mg, about 9425 mg, about 9450 mg, about 9475 mg, about 9500 mg, about9525 mg, about 9550 mg, about 9575 mg, about 9600 mg, about 9625 mg,about 9650 mg, about 9675 mg, about 9700 mg, about 9725 mg, about 9750mg, about 9775 mg, about 9800 mg, about 9825 mg, about 9850 mg, about9875 mg, about 9900 mg, about 9925 mg, about 9950 mg, about 9975 mg, orabout 10,000 mg.

In one embodiment, the salt form of 15-HETrE, 15-OHEPA or 13-HODEpresent in a composition of the invention comprises at least 90% byweight of the salt form of 15-HETrE, 15-OHEPA or 13-HODE. Compositionscontaining the salt form of 15-HETrE, 15-OHEPA or 13-HODE can compriseeven higher purity, for example at least 91% by weight, at least 92% byweight, at least 93% by weight, at least 94% by weight, at least 95% byweight, at least 96% by weight or at least 97% by weight of the saltform of 15-HETrE, 15-OHEPA or 13-HODE.

In one embodiment, the present disclosure provides a salt of a15-lipoxygenase product. In some embodiments, the salt is apharmaceutically acceptable salt. In some embodiments, the saltcomprises a lysine salt of the 15-lipoxygenase product. In someembodiments, the salt comprises a sodium salt of the 15-lipoxygenaseproduct. In some embodiments, the salt comprises an ornithine salt ofthe 15-lipoxygenase product. In some embodiments, the salt comprises apiperazine salt of the 15-lipoxygenase product. In some embodiments, thesalt comprises a meglumine salt of the 15-lipoxygenase product. In someembodiments, the salt further comprising the 15-lipoxygenase product infree acid form. In some embodiments, the salt is selected from the groupconsisting of: sodium, lysine, ornithine, piperazine, meglumine, andcombinations thereof. In some embodiments, the salt is a sodium salt. Insome embodiments, the salt is a lysine salt. In some embodiments, thesalt is ornithine. In some embodiments, the salt is a piperazine salt.In some embodiments, the salt is a meglumine salt. In some embodiments,the 15-lipoxygenase product is selected from the group consisting of:13-HODE, 15-HETrE, 15-OHEPA, 15-HETE, and combinations thereof. In someembodiments, the 15-lipoxygenase product is 13-HODE. In someembodiments, the 15-lipoxygenase product is 15-HETrE. In someembodiments, the 15-lipoxygenase product is 15-OHEPA. In someembodiments, the 15-lipoxygenase product is 15-HETE.

In some embodiments, the present disclosure provides a salt of13-hydroperoxyoctadeca-9Z,11E-dienoic acid. In some embodiments, thesalt is a sodium salt. In some embodiments, the salt is a lysine salt.In some embodiments, the salt is an ornithine salt. In some embodiments,the salt is a piperazine salt. In some embodiments, the salt is ameglumine salt.

In some embodiments, the present disclosure provides a salt of15-hydroxy-eicosa-8(Z),11(Z),13(E)-trienoic acid. In some embodiments,the salt is a sodium salt. In some embodiments, the salt is a lysinesalt. In some embodiments, the salt is an ornithine salt. In someembodiments, the salt is a piperazine salt. In some embodiments, thesalt is a meglumine salt.

In some embodiments, the present disclosure provides a salt of15-hydroxy-eicosa-5(Z),8(Z),11(Z),13(Z),17(Z)-pentaenoic acid. In someembodiments, the salt is a sodium salt. In some embodiments, the salt isa lysine salt. In some embodiments, the salt is an ornithine salt. Insome embodiments, the salt is a piperazine salt. In some embodiments,the salt is a meglumine salt.

In some embodiments, the present disclosure provides a salt of15-hydroxy-5,8,11,13-eicosatetraenoic acid. In some embodiments, thesalt is a sodium salt. In some embodiments, the salt is a lysine salt.In some embodiments, the salt is an ornithine salt. In some embodiments,the salt is a piperazine salt. In some embodiments, the salt is ameglumine salt.

In some embodiments, the present disclosure provides a compositioncomprising a salt of 13-hydroperoxyoctadeca-9Z, 11E-dienoic acid,15-hydroxy-eicosa-8(Z),11(Z),13(E)-trienoic acid, 15-hydroxy-eicosa-5(Z), 8(Z), 11(Z),13(Z),17(Z)-pentaenoic acid, and/or15-hydroxy-5,8,11,13-eicosatetraenoic acid. In some embodiments, thesalt comprises or is a sodium salt. In some embodiments, the saltcomprises or is a lysine salt. In some embodiments, the salt comprisesor is an ornithine salt. In some embodiments, the salt comprises or is apiperazine salt. In some embodiments, the salt comprises or is ameglumine salt.

In some embodiments, the present disclosure provides a pharmaceuticalcomposition comprising a salt form of a 15-lipoxygenase product. In someembodiments, the salt form of the 15-lipoxygenase product comprises thesalt of any one or more of 13-hydroperoxyoctadeca-9Z, 11E-dienoic acid,15-hydroxy-eicosa-8(Z), 11(Z),13 (E)-trienoic acid, 15-hydroxy-eicosa-5(Z),8(Z),11(Z),13(Z),17(Z)-pentaenoic acid, and/or15-hydroxy-5,8,11,13-eicosatetraenoic acid. In some embodiments, thepharmaceutical composition further comprises an excipient.

In some embodiments, after storage for at least about 4 weeks, thepharmaceutical composition comprises at least about 98%, at least about99%, or about 100% of the initial amount of the salt form of the15-lipoxygenase product. In some embodiments, after storage for at leastabout 10 weeks, the pharmaceutical composition comprises at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, at least about 99%, or about 100% of an initialamount of the salt form of the 15-lipoxygenase product. In someembodiments, after storage for at least about 24 weeks, thepharmaceutical composition comprises at least about 86%, at least about87%, at least about 88%, at least about 89%, at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, or about 100% of an initial amountof the salt form of the 15-lipoxygenase product. In any of the foregoingembodiments, the pharmaceutical composition may be stored at 2-8° C., at20° C., at 25° C., or at 40° C. In some embodiments, the pharmaceuticalcomposition is stored at 60% RH or at 75% RH.

In some embodiments, the pharmaceutical composition comprises atherapeutically effective amount of the salt of the 15-lipoxygenaseproduct. In some embodiments, the therapeutically effective amount ofthe salt form of the 15-lipoxygenase product is about 0.1 wt. % to about20 wt. %.

In some embodiments, the pharmaceutical composition is in a formsuitable for topical administration.

In some embodiments, the salt form of the 15-lipoxygenase product is thesole significant active ingredient or the sole active ingredient in thepharmaceutical composition. In other embodiments, the pharmaceuticalcomposition further comprises an additional active agent.

In some embodiments, the salt form of the 15-lipoxygenase product andthe additional active agent are co-formulated as a single dosage unit.In some embodiments, the salt form of the 15-lipoxygenase product andthe additional active agent are formulated as at least two dosage unitsfor coordinated, combined or concomitant administration.

The invention includes a therapeutic method for treating or amelioratinga disease or disorder responsive to a 15-LOX product in a subject inneed thereof comprising administering to a subject in need thereof aneffective amount of a salt form of a 15-LOX product disclosed herein.

Administration methods include administering an effective amount of acompound or composition of the invention at different times during thecourse of therapy or concurrently in a combination form. The methods ofthe invention include all known therapeutic treatment regimens.

“Effective amount” means that amount of drug substance (i.e. salt formsof 15-LOX products of the present invention) that elicits the desiredbiological response in a subject. Such response includes alleviation ofthe symptoms of the disease or disorder being treated. The effectiveamount of a disclosed salt form of a 15-LOX product in such atherapeutic method is from about 0.001 mg/kg/day to about 100 mg/kg/day,0.01 mg/kg/day to about 10 mg/kg/day, preferably from about 0.5mg/kg/day to 5 mg/kg/day.

The invention includes the use of a disclosed salt form of a 15-LOXproduct for the preparation of a composition for treating orameliorating a chronic disorder or disease or infection in a subject inneed thereof, wherein the composition comprises a mixture of one or moreof the disclosed salt forms of a 15-LOX product and an optionalpharmaceutically acceptable carrier.

“Pharmaceutically acceptable carrier” means compounds and compositionsthat are of sufficient purity and quality for use in the formulation ofa composition of the invention that, when appropriately administered toan animal or human, do not produce an adverse reaction, and that areused as a vehicle for a drug substance (i.e. salt forms of a 15-LOXproduct of the present invention). “Pharmaceutically acceptable carrier”shall also include material that (i) is compatible with the otheringredients of the composition without rendering the compositionunsuitable for its intended purpose, and (ii) is suitable for use withsubjects as provided herein without undue adverse side effects (such astoxicity, irritation, and allergic response). Side effects are “undue”when their risk outweighs the benefit provided by the composition.Non-limiting examples of pharmaceutically acceptable carriers include,without limitation, any of the standard pharmaceutical carriers such asphosphate buffered saline solutions, sterile isotonic saline, water, andemulsions such as, for example, oil/water emulsions and microemulsions.

“Pharmaceutically acceptable diluent” means compounds and compositionsthat are of sufficient purity and quality for use in the formulation ofa composition of the invention that, when appropriately administered toan animal or human, do not produce an adverse reaction, and that areused as a diluting agent for a drug substance (i.e. salt forms of a15-LOX product of the present invention).

Accordingly, in some embodiments the present disclosure provides amethod of treating a disease or disorder in subject in need thereof, themethod comprising administering to the subject a pharmaceuticalcomposition as disclosed herein. In some embodiments, the disease ordisorder is selected from the group consisting of: acne, erythema,infection, fatty liver, neuropathy, and skin inflammation. In someembodiments, the pharmaceutical composition is administered to thesubject in an amount sufficient to provide a therapeutically effectiveamount of the salt form of the 15-lipoxygenase product. In someembodiments, the therapeutically effective amount is about 0.001mg/kg/day to about 100 mg/kg/day.

In some embodiments, the present disclosure provides a method of makingthe 15-LOX compound (e.g., 13-HODE, 15-HETrE, or 15-OHEPA). In someembodiments, the method comprises contacting the corresponding 15-LOXprecursor compound (e.g., linoleic acid, DGLA or EPA) with lipoxygenase(e.g., a composition comprising lipoxygenase) in the presence of oxygen.In some embodiments, the oxygen is present at a pressure greater thanatmospheric pressure (e.g., 2-3 bar), optionally in the presence of areducing agent (e.g., cysteine) to form the 15-LOX compound. In someembodiments, a salt formation step is carried out comprising mixing asalt formation agent and the 15-LOX compound. In some embodiments thesalt forming step is carried out under oxygen-free or substantiallyoxygen-free conditions. In some embodiments the salt forming step iscarried out in the same or different vessel than the step of contactingthe 15-LOX precursor compound with lipoxygenase. In some embodiments,the method further comprises filtering the resulting 15-LOX compoundsalt. In some embodiments, the method does not include chromatographicpurification of the 15-LOX compound salt.

In some embodiments, the method comprises contacting DGLA with astoichiometric excess of lipoxygenase under a blanket of pressurizedoxygen (e.g., at about 2 bar, about 2.5 bar or about 3 bar) in thepresence of a stoichiometric excess of cysteine at a basic pH (e.g.,about 9-10) and stirred until consumption of DGLA is complete. In someembodiments, the method further comprises acidification to about pH 3-4(e.g., by adding an appropriate amount of solid citric acid) andisolating 15-HETrE free acid contained in the filtrate. In someembodiments, the 15-HETrE free acid isolated from the filtrate is thensubjected to a salt formation step, for example by exposure to asalt-forming agent such as L-lysine under oxygen-free or substantiallyoxygen free conditions in the same or separate vessel. In someembodiments, the resulting salt (e.g. lysine salt) is washed with asolvent (e.g., MtBE) once to about 4 times. In some embodiments, theresulting 15-HETrE lysine salt has a purity of at least 90%, at leastabout 92.% or at least 95% without the use of a chromatographicpurification technique.

EXAMPLES Example 1: Salts of 15-HETrE

Twenty four co-formers and acids were used to screen for stable forms of15-HETrE but most yielded oils or gels. Salts with improved handlingproperties were isolated from the screen: sodium, ornithine, lysine,meglumine and piperazine, but all were poorly crystalline. Four of thefive salts are sticky solids but the lysine salt exists as a powder andwas chemically stable at ambient temperature under vacuum for 8 days.

TABLE 1 Co-formers/acids used for salt and co-crystal screen of15-(S)-HETrE neutral acids with desirable bases (salt formers) cocrystalformers properties (oral & topical) arginine urea 4-hydroxybenzoiccholine nicotinamide ascorbic lysine cysteine azelaic (nonanedioic)meglumine allantoin benzoic ornithine — citric tromethamine (TRIS) —gallic NaHCO₃ — glycolic (hydroxyacetic) DABCO¹ — malic (L) imidazole¹ —succinic (butanedioic) piperazine — tartaric (L) ¹These co-formers arenot pharmaceutically acceptable, but were examined to see if handlingcould be improved for manufacture.

Example 2: Characterization of 15-HETrE Oil

15-HETrE was viscous oil and was confirmed by XRPD analysis to be X-rayamorphous (FIG. 1). No thermal events were observed during differentialscanning calorimetry (DSC) analysis of the material (FIG. 2). A possibleglass transition (Tg) of 15-HETrE was noted by hyper DSC analysis at−52° C. Further thermal events were noted at higher temperature buttheir causes are unknown without further analysis (FIG. 3). ¹H-NMRspectroscopy of the material showed that it was concordant withmolecular structure (FIG. 4). The FT-IR spectrum of 15-HETrE isdisplayed in FIG. 5. The carbonyl stretch of the free acid is visible at1707 cm⁻¹.

Example 3: Solubility Screen of as-Received 15-HETrE

The solubility of 15-HETrE was estimated in ten solvents using thealiquot addition method. 15-HETrE was found to be miscible in allsolvents tested, displaying high solubility in all cases. However, whencombined with 1,1,1,3,3,3-hexafluoroisopropyl acrylate (“HFIPA”), rapidcolor change from clear, colorless to blood red to deep purple/black wasobserved. This is believed to be due to degradation of 15-HETrE,therefore HFIPA was not used in any further studies of 15-HETrE. Thesolubility data are shown in Table 2 below.

TABLE 2 Estimated solubilities of 15-HETrE in a range of solvents.Solubility Range Solvent Abbreviation mg/dl Acetonitrile ACN  93-278 THF— 332-996 Acetone — 312-936 Dioxane — 166-498 DCM — 117-352 Ethylacetate EtOAc 189-566 DMF — 123-368 Ethanol EtOH 126-378 Methanol MeOH212-636

The experiments yielded gels or oils. Analysis of these materials byXRPD has shown them to be composed of X-ray amorphous materials or X-rayamorphous material and co-former. Four co-formers provided materialsthat were solid or semi-solid and possess improved handling propertiesover the starting material: NaHCO₃, ornithine, lysine and piperazine.

Example 4: 15-HETrE Sodium Salt

Material prepared according to Example 1 using NaHCO₃ and 15-HETrE wasisolated as an off white waxy solid after evaporation of theethanol/water co-solvent system. Dissolution in ethanol followed byevaporation improved the handleability of the material. Disorderedmaterial was also isolated by slurrying with MTBE.

The material was found to be composed of very disordered crystallinematerial when analyzed by XRPD (FIG. 6). Some amorphous content may alsobe present.

Peak shifting observed in the ¹H-NMR spectrum of the material suggestedsodium salt formation (FIG. 7).

FT-IR analysis of a sample of the sodium salt did not show the carbonylstretch present in the free acid at 1707 cm⁻¹, suggesting salt formation(FIG. 8).

DSC analysis of the 15-HETrE sodium salt showed a small endotherm withan onset temperature of 71.3° C. (FIG. 9). This endotherm was notobserved during the thermal analysis of the free acid (see, e.g., FIGS.2-3).

Example 5: 15-HETrE Lysine Salt

Material prepared according to Example 1 using lysine and 15-HETrE Thematerial was isolated as an off white powdery solid from the evaporationof the ethanol/water co-solvent system. Dissolution in ethanol followedby evaporation improved the handleability of the material.

The material was composed of disordered crystalline material whenanalyzed by XRPD and may contain some amorphous content (FIG. 10).Equally spaced diffraction peaks at low angle suggest the formation of amesophase such as a liquid crystal.

The same XRPD pattern was observed from a number of experimentsincluding evaporation, grinding and sonication (neat and with solvent).

Peak shifting of 15-HETrE and lysine observed in the ¹H-NMR spectrum ofthe material indicated possible salt formation (FIG. 11). The ratio of15-HETrE:lysine was 0.9:1.

FT-IR analysis of a sample of the lysine salt shows that the strongcarbonyl stretch, present in the free acid at 1707 cm⁻¹, had vanishedsuggesting salt formation (FIG. 12).

DSC analysis of the 15-HETrE lysine salt showed two small endothermswith onset temperatures of 87.1° C. and 115.5° C. respectively (FIG.13). These endotherms were not observed during the thermal analysis ofthe free acid (see, e.g., FIGS. 2-3).

Example 6: 15-HETrE Ornithine Salt

Material prepared according to Example 1 using ornithine and 15-HETrEwas isolated as an off white waxy/oily solid after evaporation of theethanol/water co-solvent system. Dissolution in ethanol followed byevaporation improved the handleability of the material. The material wascomposed of very disordered crystalline material when analyzed by XRPD,and may contain some amorphous content (FIG. 14).

The same XRPD pattern was observed from a number of experimentsincluding evaporation, grinding, precipitation and sonication (neat andwith solvent).

Peak shifting of both 15-HETrE and ornithine was observed in the 1H-NMRspectrum indicating probable salt formation (FIG. 15). The ratio ofHETrE:ornithine was measured as 1:0.8.

FT-IR analysis of a sample of the ornithine salt shows that the strongcarbonyl stretch, present in the free acid at 1707 cm⁻¹, had vanishedsuggesting salt formation (FIG. 16).

DSC analysis of the 15-HETrE lysine salt showed two endotherms withonset temperatures of 34.7° C. and ˜120° C. respectively (FIG. 17). Thefirst endotherm may be due to the melt of the material while the secondlarger endotherm may be due to decomposition of the material. Theseendotherms were not observed during the thermal analysis of the freeacid (see, e.g, FIGS. 2-3).

Example 7: HETrE Piperazine Salt

Yellow, semi-solid material was isolated from the sonication of neatpiperazine and 15-HETrE, followed by evaporation of the 1:1 isobutylacetate and ethanol co-solvent system.

XRPD analysis showed that the salt was composed of very disorderedcrystalline material (FIG. 18). Some amorphous content may also bepresent.

Peak shifting was observed in the ¹H-NMR spectrum of the suspected saltsuggesting salt formation (FIG. 19). The ratio of 15-HETrE:piperazinewas 1:0.66.

Example 8: Properties of Sodium, Lysine, Ornithine and Piperazine Saltsof 15-HETrE

Properties of 15-HETrE salts prepared according to Examples 4-7 werecompared against each other and against 15-HETrE free acid API. Thesodium, ornithine and piperazine salts were each sticky or waxy solids,but the lysine salt could be made as a powder and was chemically stableat ambient temperature under vacuum for 8 days.

Table 3 compares the properties of each salt that were evaluated duringthe screen and includes crystallinity, appearance, solvent content byNMR, chemical stability, preparation method and stoichiometry by NMRanalysis.

The lysine salt represents the best candidate for development as itcould be prepared as a handleable powder and was made by a variety ofmethods. Salt formation was confirmed by ¹H-NMR and FT-IR spectroscopyand the salt exhibited an equimolar stoichiometry.

TABLE 3 Table of properties of solids isolated from the salt/cocrystalscreen Solvent 8 Day Stability Stoichiometry Compound CrystallinityAppearance Content 40/75% RH Prep method (base:API) HETrE Very Off whiteTrace THF or 97% purity Evaporation,   1:1 lysine salt disordered powderEtOH slurry, sonication HETrE Very Off white trace EtOH 95% purityEvaporation Cannot be determined sodium salt disordered waxy solid orslurry by NMR HETrE Very Off white Trace THF or 96% purity Evaporation,0.9:1 ornithine salt disordered waxy solid, EtOH slurry, yellowsonication coloration on storage HETrE Very Yellow sticky Trace EtOH Nottested Evaporation 0.7:1 piperazine salt disordered semi-solid orsonication HETrE API amorphous oil — — — —

The sodium salt was used for further study as a backup candidate as itwas a waxy solid and was made by a number of different methods.Stoichiometry could not be determined by NMR spectroscopy.

The ornithine salt was not recommended for further study as it was quitesticky and displayed a yellow coloration over time. The piperazine saltexhibited the poorest handling properties and was a sticky, semi-solidmaterial that appears to flow on exposure to air at ambient temperature,probably due to uptake of moisture.

Photomicroscopic images of each salt are displayed in FIGS. 20A-H.Images of each salt were captured under crossed-polarized light with andwithout a 1st order red filter, which enhances contrast betweencrystalline and amorphous content. The length of the scale barrepresents 200 μm. The bright colors are evidence of crystallinity.

Example 9: 15-HETrE Lysine Salt Scale Up

A slurry method was found as an alternative to the previously usedrotary evaporation method to prepare the lysine salt using EtOAc assolvent, but failed to generate the salt when scaled up. Addition of 9%MeOH to EtOAc generated the salt on a 800 mg scale, with a purity of96%. Stability studies have commenced for the lysine salt (20° C./60% RHair/N₂ and 40° C./75% RH air/N₂, open/closed vials).

An alternative method to prepare 15-HETrE sodium salt by slurrying andavoid need for rotary evaporation could not be found. If water was used,the base dissolved but the resulting salt was also highly soluble andthus did not precipitate. If water was excluded, base did not dissolveand therefore did not react with 15-HETrE. Scale up and characterizationwas completed for 15-HETrE sodium salt using rotary evaporation from 10%aqueous EtOH, then EtOH. The recovered material had a yellow coloration.Purity as measured by UPLC analysis was ˜81%.

Example 10: Characterization of the Scaled Up 15-HETrE-Lysine Salt

15-HETrE-Lysine salt prepared according to Example 9 was analyzed byXRPD and found to consist of disordered crystalline material (FIG. 21).

Salt formation was confirmed by ¹H-NMR spectroscopy (solvent: d₄-MeOD),as evidenced by peak shifting of both lysine and 14-HETrE protons (FIG.22). The ratio of HETrE:lysine was measured as 1:1.

TG/DTA analysis of the 15-HETrE-Lysine salt exhibited negligible weightloss<160° C. indicating that the salt was anhydrous. A small endothermat 116° C. (onset) was noted but the cause is unknown (FIG. 23).

The DSC thermogram displayed a number of small endotherms with onsets at88° C. and 115° C., followed by broader endotherms, which may beassociated with melt and decomposition events (FIG. 24).

Hot stage microscopy of the salt between 30° C. and 200° C. showed noobservable melt until approximately 140° C., but the material had notcompletely melted at 200° C. (FIGS. 25A-D).

Hyper DSC analysis of the material indicated that the glass transition(T_(g)) temperature was 16° C. (half C_(p) value, FIG. 26).

DVS analysis of the 15-HETrE lysine salt showed less than 1% weight gainbelow 70% RH but gained an additional 1% between 70-80% RH (FIG. 26) andindicates that the material was hygroscopic according to the EuropeanPharmacopeia classification. The weight gain above 80% RH wassufficiently large to suggest that the sample deliquesced. Post DVS XRPDanalysis of the sample showed that it was consistent with that of theinput material (FIG. 21).

FIG. 27 shows the FT-IR spectrum of the 15-HETrE lysine salt preparedaccording to Example 9.

Example 11: Scale Up and Operability Study of 15-HETrE Lysine Salt

Initially the 15-HETrE lysine salt was isolated by rotary evaporation inorder to remove residual solvent and reduce stickiness of the solids.However, a small number of experiments were carried out to find a moresuitable method that could be used to prepare the salts as dry solids ona larger scale.

Slurry experiments were set up to form the lysine salt under differentconditions. Solvents were added according to Table 4 below, Solvent:antisolvent ratios were 1:3.

TABLE 4 Slurry experiments to prepare HETrE lysine salt Solvent/ SampleNo. antisolvent Observations XRPD 1946-035-01 EtOAc off white powdersame as previous lysine salts 1946-035-02 Acetone- off white powder sameas previous EtOAc lysine salts, some new peaks 1946-035-03 EtOH- offwhite powder with same as previous MTBE yellow waxy solid lysine salts1946-035-04 IPA-EtOAc off white powder with same as previous yellow waxysolid lysine salts 1946-043-01 EtOAc white solid in solution same asprevious lysine salts 1946-047-01 EtOAc yellow/peach solid HETrE lysinesalt + free lysine 1946-055-01 EtOAc white/cream solid HETrE lysine salt1946-055-02 EtOAc-EtOH white/cream solid HETrE lysine salt + free lysine1946-055-03 EtOAc- white/cream solid HETrE lysine salt acetone1946-055-04 EtOAc-IPA white/cream solid HETrE lysine salt 1946-055-05EtOAc- white/cream solid HETrE lysine salt MeOH 1946-059-01 EtOAc cloudysolution N/A 1946-059-02 EtOH white/cream solid HETrE lysine salt1946-059-03 10:1 EtOAc: white/cream solid HETrE lysine salt MeOH1946-059-04 Acetone yellow/brown solution N/A 1946-059-05 IPAwhite/cream solid HETrE lysine salt 1946-063-01 10:1 EtOAc: pale yellowsolid HETrE lysine salt MeOH 1946-067-01 10:1 EtOAc: pale yellow solidHETrE lysine salt MeOH

The 15-HETrE lysine slurries yielded off white powders with some waxyyellow solids. The XRPD pattern matched previous 15-HETrE lysine saltpatterns. Sample 1946-035-01 in EtOAc was selected for scale up to 100mg, followed by 1 g scale.

The material scaled up successfully at 100 mg, however, issues wereencountered when preparing the salt on a 1 g scale.

The reaction took much longer than previously observed to go tocompletion (6 days vs. 1 day).

The material appeared to be very fine and could not be easily filtered.The 15-HETrE lysine salt was isolated via centrifugation, decanted anddried under vacuum. The material was isolated as a yellow solid but wascontaminated with residual lysine.

Further experiments were conducted to find an alternative solvent andEtOAc-MeOH (9:1) was found to be most suitable as salt formationoccurred within 1 hour of addition. Other solvent combinations generatedthe salt but required several hours or overnight mixing. Scale up on 250mg then 800 mg scales were successful and salt was formed within severalhours, filtered and dried.

Purity by UPLC was 96% for the 800 mg scale batch and 97% for the 250 mgscale batch.

FIG. 28 shows the stability of the 15-HETrE-lysine salt compared to15-HETrE free fatty acid at various conditions. Free 15-HETrE isunstable and degrades at all conditions with the exception of −20° C.The 15-HETrE-lysine salt is stable at 20° C. and 40% humidity as well asat 40° C. and 75% humidity, making the lysine salt useful forpharmaceutical applications. 15-HETrE lysine salt was prepared on alarger scale for the crystallization screen as a white powder. XRPDanalysis indicated that it was disordered and was very similar toprevious analyses.

TABLE 5 Crystallization screen of 15-HETrE lysine salt Sample No.Solvent/ (1922_) antisolvent Conditions Observatons 077-01 EtOH-waterRotary evap Large scale prep 087-01 1:1:1 MeOH: evaporation off whitepowder ACN:Dioxane 087-02 20:1 evaporation off white powder, slightlysticky Dioxane:water powder, some birefringence 087-03 3:1:3 MIBK:evaporation orange tinged powdery solid, Acetone:MeOH slightly vstickywith clumps, little birefringence 091-01 EtOAc vapor stress slightlysticky powder, minor birefringence 091-02 acetone vapor stress slightlysticky powder, minor birefringence 091-03 DCM vapor stress slightlysticky powder, minor birefringence 083-01 MTBE slurry (RT) off whitewaxy solid. Mesophase, birefringence film 083-02 isobutyl acetate slurry(RT) white paste 083-03 cyclohexane slurry (RT) white paste-mesophase.Film, some birefringence 083-02A Et₂O/ MTBE slurry (RT) white solid083-03A EtOH/ EtOAc slurry (RT) Birefringent gel with white solid083-03A — Vac drying birefringent white sticky solid 083-04 Et₂O slurry(RT) yellow waxy solid. Mesophase. Spherulites. Film, some birefringence083-04A Dioxane/ EtOAc slurry (RT) Yellow sticky solid + solution083-04A — Vac drying Birefringent yellow solid 087-01A ACN/ EtOAc slurry(RT) pale yellow gel 087-02A Acetone/ EtOAc slurry (RT) pale yellow gelwith solid 087-02A — Vac drying pale yellow solid 087-03A Cyclohexane/slurry (RT) pale yellow solid with solution EtOAc 087-03A — vac dryingpale yellow solid 091-01A DCM/ EtOAc slurry (RT) pale yellow solid withsolution 091-02A MeOH/ EtOAc slurry (RT) pale yellow solid with somecrystals 091-03A Et₂O/ EtOAc slurry (RT) white solid in solution

Example 12: Scale Up and Operability Study of 15-HETrE Sodium Salt

The 15-HETrE sodium salt was a highly disordered by XRPD analysis (FIG.9) and was a waxy solid.

Analysis of the material by ¹H-NMR spectroscopy (solvent: d₄-MeOD),indicated salt formation had occurred, as evidenced by peak shifting of15-HETrE protons (FIG. 10). Stoichiometry could not be determined by¹H-NMR spectroscopy.

TG/DTA analysis of the 15-HETrE sodium salt displayed a continuousgradual weight loss above ambient temperature, which is not unusual forvery disordered materials and is probably due to loss of volatilecomponents.

DSC thermogram displayed a complex series of small thermal events, thecauses of which are unknown.

A distinct glass transition (T_(g)) signal was not observed from thehyper DSC thermogram.

DVS analysis of the 15-HETrE-sodium salt showed that the sample gainedless than 1% weight below 40% RH but exponentially increased in weightgain thereafter and deliquesced at high RH. This indicates that thematerial is very hygroscopic according to the European Pharmacopeiaclassifications. Post DVS XRPD analysis was not conducted as thematerial deliquesced.

FIG. 15 shows the FT-IR spectrum for 15-HETrE sodium salt preparedaccording to this Example.

Initially the 15-HETrE sodium salt was isolated by rotary evaporation inorder to remove residual solvent and reduce stickiness of the solids.However, a small number of experiments were carried out to find a moresuitable method that could be used to prepare the salts as dry solids ona larger scale.

Slurry experiments were set up to form the sodium salt under differentconditions. Solvents were added according to the table below, Solvent:antisolvent ratios were 1:3.

For non-aqueous slurries, the base was added as a solid but did notdissolve. Addition of base as an aqueous solution was also tried butprecipitation of salt did not occur and solvent had to be evaporated.Therefore, a suitable slurry method was not found to prepare HETrEsodium salt.

Scale up was carried out using rotary evaporation on a gram scale andgenerated the salt but the solid exhibited a yellow coloration.

Purity as measured by UPLC was ˜81%.

TABLE 6 Attempts to prepare 15-HETrE sodium salt by a slurry methodSolvent/ Sample No. antisolvent Observations 1946-035-05 EtOAc¹ cloudysolution 1946-035-06 Acetone- cloudy solution EtOAc¹ 1946-035-07EtOH-MTBE¹ cloudy solution 1946-035-08 IPA-EtOAc¹ cloudy solution1946-035-09 MTBE-EtOH cloudy solution 1946-035-10 heptane-EtOH cloudysolution 1946-035-11 cyclohexane- cloudy solution EtOH 1946-035-12IPOAc-EtOH cloudy solution 1946-035-05A acetone solution, not dissolved1946-035-06A ACN solution, not dissolved, yellow solid 1946-035-07A DMFcloudy yellow solution, not dissolved 1946-035-08A DMSO pale yellowsolution, not dissolved 1946-035-09A EtOH cloudy solution, not dissolved1946-035-10A IPA cloudy solution, not dissolved 1946-035-11A MeOH cloudysolution, not dissolved 1946-035-12A THF cloudy solution, not dissolved1946-035-05B Acetone-H₂O solution, pale yellow, not dissolved1946-035-12B THF-H₂O solution, pale yellow, not dissolved 1946-045-01Acetone² solution, yellow 1946-045-02 THF² solution, slightly yellowNote¹: 560 μL additional MTBE added to try form precipitate. Note²:saturated aqueous NaHCO3 was added to slurry.

TABLE 7 Crystallization screen of HETrE sodium salt Sample No. (1922-)Solvent/antisolvent Conditions Observations 075-01 EtOH-water Rotaryevap Large scale prep 085-01 1:1:1 MeOH: evaporation mesophase.Spherulites, ACN:Dioxane slightly sticky particles, some birefingence085-03 3:1:1 MIBK: evaporation mesophase - looks like Acetone:MeOH smallneedles in mesophase 085-02 20:1 Dioxane:water evaporation mesophase,slightly sticky particles, some birefringence 089-01 EtOAc vapourstresss lightly sticky with some birefringence, powder 089-02 acetonevapour stresss lightly sticky with some birefringence 089-03 DCM vapourstresss lightly sticky with some birefringence 079-01 MTBE slurry (RT)off white waxy solid - bright colors, birefringence film 079-02 Isobutylacetate Slurry (RT) white gel, paste bright colors 079-02A EtOH-MTBEslurry (RT) white gel 079-03 cyclohexane slurry (RT) white paste atbottom. Solid round sides, sticky powder 079-03A Dioxane-MTBE slurry(RT) yellow solid, gel 079-04 Et₂O slurry (RT) white/clear waxy solid,birefringent, birefringence film 085-01A ACN-MTBE slurry (RT)Birefringent waxy solid 085-01A — vacuum birefringent waxy drying solid085-02A Acetone-MTBE slurry (RT) Birefringent waxy solid 085-02A —vacuum birefringent waxy drying solid 089-01A Cyclohexane- slurry (RT)Birefringent, waxy MTBE solid off-white 089-01A — vacuum Birefringent,waxy drying solid off-white 089-02A DCM- MTBE slurry (RT) Birefringent,waxy white solid 089-02A — vacuum Birefringent, waxy drying white solid089-03A MeOH- MTBE slurry (RT) Yellow sticky solid, some Birefringence089-03A — vacuum Yellow sticky solid, drying some Birefringence

Example 13: Scale Up and Operability Study of 15-HETrE Sodium MeglumineSalt

A sample prepared from 15-HETrE with N-methyl-D-glucamine (meglumine)yielded a gel and was stored under refrigerated conditions to encouragecrystallization. After some time, it was noticed that the material hadsolidified and the XRPD pattern indicated presence of crystallinity,although there was some amorphous content as well (FIG. 29). The XRPDpattern was different to that of meglumine indicating formation of aunique solid form.

Proton NMR spectrum of the sample indicated a 1:1 stoichiometry and peakshifting was noted indicating salt formation (FIG. 30). One mole of EtOHwas detected, which may indicate solvate formation.

Although the XRPD pattern indicated that it was the most crystallinesample generated from the screen, the sample was a sticky solid andflowed when pressed between microscope slides indicating liquidcrystalline properties (FIGS. 31A-B). The scale bar in FIGS. 31A-Brepresents 200 μm. The material was dried overnight under vacuum toremove residual EtOH and the sample was still sticky.

15-HETrE meglumine salt was prepared on a larger scale for thecrystallization screen as a viscous oil.

The meglumine salt was then subdivided into multiple vials and subjectedto different stress conditions and samples purged with nitrogen (Table8). Aqueous and non-aqueous conditions were employed and solvents driedusing molecular sieves. Solvents were also purged with nitrogen.

Most of the samples remained gels when stressed under differentconditions. However, one sample partially crystallized in the fridgefrom ACN-EtOH. The sample was used for seeding other samples but nofurther crystallization was noted.

TABLE 8 Crystallization screen for 15-HETrE meglumine salt Solvent/Sample No. antisolvent Conditions Observations 1922-073-01 EtOH-waterRotary evap Large scale prep 1922-081-09 — 40° C./75%RH dark yellow gel1922-095-06 — temp. stress pale yellow gel 1922-093-01 EtOAc- vaporstress solution EtOH 1922-093-01 — vacuum drying pale yellow gel1922-093-02 acetone- vapor stress Solution. EtOH 1922-093-02 — vacuumdrying pale yellow gel 1922-093-03 DCM-EtOH vapor stress solution1922-093-03 — vacuum drying pale yellow gel 1922-093-04 ACN-EtOH vaporstress white solid, birefringent needles. Melted when removed fromfridge. Used as seeds. Solidified in fridge to white solid, sticky,agglomerates 1922-093-05 THF-EtOH vapor stress solution 1922-093-05 —vacuum drying pale yellow gel 1922-093-10 water-EtOH vapor stresssolution 1922-093-10 — vacuum drying pale yellow gel 1922-081-07EtOH-water slurry (RT) solution 1922-081-08 EtOH: slurry (RT) solutionIPOAc 1946-003-01 water-EtOH slurry (RT) solution 1946-003-01 — vacuumdrying pale yellow gel 1946-003-02 IPOAc- slurry (RT) solution EtOH1946-003-02 — vacuum drying pale yellow gel 1946-003-03 ACN-MTBE slurry(RT) solution 1946-003-03 — vac drying pale yellow gel 1946-003-04acetone- slurry (RT) gel. Seeded with MTBE 1922-093-04 1946-003-04A —seeding yellow gel with seed 1946-003-05 cyclohexane- slurry (RT) gel.Seeded with MTBE 1922-093-04 1946-003-05A — seeding yellow gel with seed1946-003-06 1946-003-06 — vac drying pale yellow gel 1946-003-07dioxane- slurry (RT) Solution EtOAc 1946-003-07 — vac drying pale yellowgel

Based on these results, 15-HETrE lysine and sodium salts still representthe best candidates for further study as they are less sticky than themeglumine salt. However, the properties of the meglumine salt may showimprovement with further processing.

Example 14: Preparation of 15-(S)-HETrE from DGLA

Sodium borate buffer (0.1 M) was prepared by charging boric acid (61.8g, 1 mol) and NaOH (120.0 g, 3 mol) in water (10 L). A 20 L hastealloyvessel was charged with 10.0 L of the sodium borate buffer, followed bycysteine (237.2 g, 1.958 mol, 2.0 equiv). After the buffer and cysteinewere fully dissolved with stirring, DGLA (300 g, 9.79 mol) was added andthe mixture was cooled to 0-5° C. at pH 9.6. LPX1 enzyme powder (2.66 g,8.85 mg/g DGLA, 1.8 Munits/g DGLA, 0.88 wt. %) was added and the vesselwas pressurized to 2.5 bar with pure oxygen. After stirring for onehour, the oxygen head pressure was slowly released to avoid foaming. Analiquot was removed and acidified to pH 3 with citric acid solution (25%w/v). The aliquot was extracted with deuterated chloroform, dried oversodium sulfate, filtered and analyzed by NMR.

Additional LPX1 enzyme powder (0.503 g, 1.8 mg/g DGLA, 0.36 Munits/gDGLA, 0.18 wt. %) and one additional equivalent of cysteine (117.0 g,0.979 mol) were added and the mixture was stirred under oxygen foranother hour. Analysis of another aliquot as described above indicatedthat the reaction was complete.

The mixture was purged with nitrogen (2 bar, 2 cycles) and stirred at900 rpm overnight under a blanket of nitrogen. The mixture was thencharged to a nitrogen-blanketed 10 L jerry can. The mixture was thenadded to a 20 L reactor under a blanket of argon and solid citric acidwas added in 50 g portions until the pH dropped to 3.5.

Precipitated solid was collected with a sintered funnel under argon.After transferring the solids back to the reactor, 3 L of methyl t-butylether (MtBE) were added and the mixture was stirred at 400 rpm for 10minutes. These steps were repeated twice more before residual solventwas evaporated at 250 mbar with a 40° C. bath (rotovap) until no moredistillate was observed. The rotovap was vented with argon.

Example 15: Purification of Crude 15-(S)-HETrE by Chromatography

Half of the crude 15-(S)-HETrE prepared in Example 14 was dissolved in130 mL of a MtBE/cyclohexane (20:80 v/v) solvent mixture. The solutionwas divided into two portions; each portion was purified using a Biotage75L silica cartridge which had been pre-eluted with cyclohexane. Elutionof the 15-(S)-HETrE was accomplished in 5 stages: (1) MtBE/cyclohexane(10:90): 4 L; (2) MtBE/cyclohexane (20:80): 3 L; (3) MtBE/cyclohexane(30:70): 2 L; (4) MtBE/cyclohexane (50:50): 4 L; and (5) MtBE: 2 L.Fractions including 15-(S)-HETrE by TLC analysis (fractions 4-9) werecombined and concentrated by rotovap (250 mbar at 40° C.), under argonuntil no more distillate was observed. Purity was 95.5% (by ¹H-NMR) or96.4% (by uHPLC).

Example 16: Preparation of 15-(S)-HETrE L-Lysine Salt

L-Lysine mono-hydrate (399±2 mg) was suspended in alcohol (Table 9) in a20 mL snap cap vial, 15-(S)-HETrE (803±2 mg) was dissolved in thenon-polar solvent (4 mL) and added to the stirring suspension of lysine,further non-polar solvent (2 mL) was used to rinse the HETrE vial andadded to the lysine/HETrE suspension. The balance of the non-polarsolvent was then added (total volume of solvent added was 8 mL), seedwas added at this point for experiment 3 to 10, and stirred at 250 rpmovernight.

TABLE 9 Solvent systems. Reaction No. Solvent (non-polar) Solvent(polar) ratio 2032-001-1  Ethyl acetate methanol 95:5  2032-001-2  Ethylacetate methanol 90:10 2032-001-3  Ethyl acetate ethanol 90:102032-001-4  Ethyl acetate Isopropyl alcohol 80:20 2032-001-5  Ethylacetate acetone 80:20 2032-001-6  Iso-propyl acetate methanol 95:5 2032-001-7  Iso-propyl acetate methanol 90:10 2032-001-8  Iso-propylacetate ethanol 90:10 2032-001-9  Iso-propyl acetate Isopropyl alcohol80:20 2032-001-10 Iso-propyl acetate acetone 80:20

Reaction Nos. 1 and 2 were not seeded; Reaction Nos. 3 to 10 were seededwith seeds obtained from the filter cake of Reaction No. 1.

Products of each of the Reactions were white in appearance. Filtrationwas carried out with a sintered funnel (no. 3, 10 mm diameter) undermild vacuum. Some filter cake products were yellow in appearance. Thefirst ˜200 μL of filtrate were transferred into an HPLC vial. Filtrationof the remaining reaction mixture (alternating between high and mediumvacuum) yielded a filter cake that was transferred to a vacuum chamberand dried overnight before being stored in a freezer. The salt itself isinsoluble in chloroform and DMSO, but dissolves readily in water.

NMR analysis in D₂O was used to determine lysine content of the salt(Table 10) by calibrating the alpha proton of lysine to 1.

TABLE 10 Lysine content of 15(S)-HETrE L-lysine salts. Reaction No.Lysine signal HEtrE at 5.6 ppm difference HETrE at 6 ppm difference2032-001-1  1 0.9493 0.01941 0.94 0.00698 2032-001-2  1 0.9095 −0.020390.9164 −0.01662 2032-001-3  1 0.9277 −0.00219 0.9622 0.02918 2032-001-4 1 0.9283 −0.00159 0.9237 −0.00932 2032-001-5  1 0.9488 0.01891 0.93710.00408 2032-001-6  1 0.9503 0.02041 0.9451 0.01208 2032-001-7  1 0.9247−0.00519 0.9115 −0.02152 2032-001-8  1 0.9032 −0.02669 0.9434 0.010382032-001-9  1 0.9407 0.01081 0.9311 −0.00192 2032-001-10 1 0.9164−0.01349 0.9197 −0.01332 average = 0.92989 0.93302

Observations of the Reactions are summarized in Table 11.

TABLE 11 Filtration Reaction Cake weight Cake purity Filtrate puritytime approx. Filter cake Slurry No. Solvent ratio (mg) (%) (%) (mins)appearance mobility Drainage 2032-001-1 Ethyl 95:5 921 97.1 83.05 35 Y,H 4 3 acetate/methanol 2032-001-2 Ethyl 90:10 990 97.1 82.77 30 W, P 4 3acetate/methanol 2032-001-3 Ethyl 90:10 945 97.1 82.61 25 Y, H 4 3acetate/ethanol 2032-001-4 Ethyl 80:20 813 97.1 82.13 30 Y, H 4 3acetate/Isopropyl alcohol 2032-001-5 Ethyl 80:20 1113 96.8 — 50 Y, H 5 4acetate/acetone 2032-001-6 isopropyl 95:5 932 97.2 86.91 30 Y, H 4 3acetate/methanol 2032-001-7 isopropyl 90:10 1000 96.8 81.04 35 W, P 4 3acetate/methanol 2032-001-8 isopropyl 90:10 951 97.1 83.39 35 Y, H 4 3acetate/ethanol 2032-001-9 isopropyl 80:20 932 96.9 80.24 35 Y, H 4 3acetate/Isopropyl alcohol 2032-001-10 isopropyl 80:20 910 96.8 79.64 50Y, H 5 4 acetate/acetone Filtration time: time for cake to reach semidry state, not wet Filter cake appearance: appearance of cake afterdrying, w = white, y = yellow (colour of cake), H = hard, P = powderySlurry mobility: 1 = thin, very mobile, 2 = medium thick, mobile, 3 =thick but transfer mostly prior to rinsing, 4 = thick not easilytransferable, 5 = very thick, no flow without fresh diluent Drainage(suction filtration): 1 = easily, 2 = medium, 3 = difficult, 4 = none

The impurity profile of Reaction Nos. 1 to 10 are shown in Table 12.

TABLE 12 Impurity profiles retention peak area 001-1 001-1 001-2 001-2001-3 001-3 RRT time (%) (salt) (liquor) (salt) (liquor) (salt) (liquor)IMP 1 0.62 4.35 0.13 0.03 0.35 0.02 0.25 0.02 0.31 IMP 2 0.64 4.44 0.190.05 0.5 0.04 0.36 0.04 0.47 IMP 3 0.69 4.81 0.31 0.07 0.78 0.05 0.560.06 0.71 IMP 4 0.91 6.34 1.22 0.95 2.9 1.01 2.61 0.99 3.23 HETrE 1 6.9797.3 97.07 83.05 97.09 82.77 97.11 82.61 IMP 5 1.03 7.2 0 0.11 0.8 0.10.89 0.12 1.09 IMP 6 1.1 7.7 1.5 1.48 1.54 1.45 1.54 1.48 1.57 IMP 71.75 12.25 unknown 0.1 2.87 0.1 3.3 0.11 3.56 retention peak area 001-4001-4 001-5 001-5 001-6 001-6 RRT time (%) (salt) (liquor) (salt)(liquor) (salt) (liquor) IMP 1 0.62 4.35 0.13 0.03 0.42 0.03 — 0.03 0.19IMP 2 0.64 4.44 0.19 0.04 0.51 0.05 — 0.04 0.27 IMP 3 0.69 4.81 0.310.06 0.86 0.07 — 0.06 0.43 IMP 4 0.91 6.34 1.22 0.99 3.59 0.99 — 0.962.32 HETrE 1 6.97 97.3 97.07 82.13 96.77 — 97.17 86.91 IMP 5 1.03 7.2 00.12 1.17 0.12 — 0.12 0.62 IMP 6 1.1 7.7 1.5 1.47 1.7 1.46 — 1.42 1.67IMP 7 1.75 12.25 unknown 0.13 3.77 0.18 — 0.12 2.15 retention peak area001-7 001-7 001-8 001-8 001-9 001-9 RRT time (%) (salt) (liquor) (salt)(liquor) (salt) (liquor) IMP 1 0.62 4.35 0.13 0.03 0.4 0.02 0.33 0.020.43 IMP 2 0.64 4.44 0.19 0.05 0.43 0.04 0.51 0.05 0.64 IMP 3 0.69 4.810.31 0.05 0.67 0.05 0.75 0.06 0.92 IMP 4 0.91 6.34 1.22 1.07 2.65 0.962.9 1.01 3.07 HETrE 1 6.97 97.3 96.83 81.04 97.1 83.39 96.92 80.24 IMP 51.03 7.2 0 0.12 1.17 0.12 0.86 0.12 1.06 IMP 6 1.1 7.7 1.5 1.48 1.621.44 1.62 1.48 1.6 IMP 7 1.75 12.25 unknown 0.13 4.08 0.11 3.36 0.134.08 retention peak area 001-10 001-10 RRT time (%) (salt) (liquor) IMP1 0.62 4.35 0.13 0.03 0.42 IMP 2 0.64 4.44 0.19 0.07 0.59 IMP 3 0.694.81 0.31 0.07 0.82 IMP 4 0.91 6.34 1.22 0.99 3.1 HETrE 1 6.97 97.3 96.879.64 IMP 5 1.03 7.2 0 0.11 1.01 IMP 6 1.1 7.7 1.5 1.46 1.68 IMP 7 1.7512.25 unknown 0.18 3.43

Example 17: Investigation of Methanol as Co-Solvent

Based on the results of Example 16, ratios of methanol with ethylacetate or isopropyl acetate were investigated to determine effects on15-(S)-HETrE yield, as shown in Table 13.

TABLE 13 Solvent Exp. No. (non-polar) Solvent (polar) ratio 009-1 Ethylacetate methanol 90:10 009-2 Ethyl acetate methanol 85:15 009-3 Ethylacetate methanol 80:20 009-4 Iso-propyl acetate methanol 90:10 009-5Iso-propyl acetate methanol 85:15 009-6 Iso-propyl acetate methanol80:20

Results of the reactions are summarized in Table 14.

TABLE 14 Cake Cake Filtrate Filtration weight purity purity time approx.Filter cake Slurry Exp. No. (mgs) (%) (%) (mins) appearance mobilityDrainage 009-1  876* 96.69 78.09 30 W, P 4 3 009-2 996 96.73 79.28 35 W,P 4 3 009-3 999 96.66 77.39 35 W, P 4 3 009-4 943 96.66 79.23 30 W, P 43 009-5 1000  96.41 79.38 35 W, P 4 3 009-6 987 96.51 76.69 30 W, P# 4 3*= reaction filtered through a larger filter initially but transferredto smaller sinter funnel. #= cake had yellow hard characteristics onedge of cake. Filtration time: time for cake to reach semi dry state,not wet Filter cake appearance: appearance of cake after drying, w =white, y = yellow (colour of cake), H = hard, P = powdery Slurrymobility: 1 = thin, very mobile, 2 = medium thick, mobile, 3 = thick buttransfer mostly prior to rinsing, 4 = thick not easily transferable, 5 =very thick, no flow without fresh diluent Drainage (suction filtation):1 = easily, 2 = medium, 3 = difficult, 4 = none

Impurity profiles of the Lysine salt and liquor portions of theexperiments are summarized in Table 15.

TABLE 15 Impurity profiles. retention peak area 009-1 009-1 009-2 009-2009-3 009-3 RRT time (%) (salt) (liquor) (salt) (liquor) (salt) (liquor)IMP 1 0.62 4.35 0.13 0.04 0.15 0.03 0.17 0.04 0.19 IMP 2 0.64 4.44 0.190.09 0.49 0.09 0.49 0.09 0.52 IMP 3 0.69 4.81 0.31 0.11 1.13 0.11 1.140.11 1.23 IMP 4 0.91 6.34 1.22 1.04 2.46 0.98 2.55 1.01 2.52 HETrE 16.97 97.3 96.69 78.09 96.73 79.28 96.66 77.39 IMP 5 1.03 7.2 0 0.14 0.850.14 0.88 0.15 0.87 IMP 6 1.1 7.7 1.5 1.42 1.36 1.39 1.47 1.39 1.37 IMP7 1.75 12.25 unknown 0.23 4.44 0.25 4.24 0.26 4.45 retention peak area009-4 009-4 009-5 009-5 009-6 009-6 RRT time (%) (salt) (liquor) (salt)(liquor) (salt) (liquor) IMP 1 0.62 4.35 0.13 0.04 0.22 0.05 0.23 0.030.32 IMP 2 0.64 4.44 0.19 0.13 0.49 0.13 0.51 0.04 0.52 IMP 3 0.69 4.810.31 0.14 1.1 0.07 1.17 0.14 1.18 IMP 4 0.91 6.34 1.22 1.01 2.54 0.992.57 1.06 2.61 HETrE 1 6.97 97.3 96.66 79.23 96.41 79.38 96.51 76.69 IMP5 1.03 7.2 0 0.14 0.89 0.12 0.84 0.14 1.14 IMP 6 1.1 7.7 1.5 1.39 1.561.46 1.5 1.39 1.46 IMP 7 1.75 12.25 unknown 0.2 4.43 0.18 4.28 0.31 4.84

Example 18: Ratio of Lysine to 15-(S)-HETrE

The effect of the ratio of lysine to 15-(S)-HETrE was investigated usingthe protocol of Example 16. Initial ratios of lysine and 15-(S)-HETrEused are shown in Table 16.

TABLE 16 Reaction Solvent Solvent Lysine No. (non-polar) (polar) Ratio(mol %) 013-1 Ethyl acetate Methanol 85:15 97.5 013-2 Ethyl acetateMethanol 85:15 95 013-3 Isopropyl acetate Methanol 85:15 97.5 013-4Isopropyl acetate Methanol 85:15 95 013-5 Ethyl acetate Methanol 80:2097.5 013-6 Ethyl acetate Methanol 80:20 95

Results of the six reactions are summarized in Table 17.

TABLE 17 Filtration Reaction Cake weight Cake purity Filtrate timeapprox. Filter cake Slurry No. (mg) (%) purity (%) (min) appearancemobility Drainage 013-1 972 96.43 85.66 30 W, P 4 3 013-2 975 96.6786.41 35 W, P 4 3 013-3 1011 96.83 86.73 35 W, P 4 3 013-4 926 96.6187.31 35 W, P 4 3 013-5 934 96.61 86.00 30 W, P 4 3 013-6 827 96.4785.85 35 W, P 4 3 Filtration time: time for cake to reach semi drystate, not wet Filter cake appearance: appearance of cake after drying,w = white, y = yellow (colour of cake), H = hard, P = powdery Slurrymobility: 1 = thin, very mobile, 2 = medium thick, mobile, 3 = thick buttransfer mostly prior to rinsing, 4 = thick not easily transferable, 5 =very thick, no flow without fresh diluent

The final ratio of lysine to 15-(S)-HETrE was determined by NMR, asshown in Table 18.

TABLE 18 Reaction lysine peak at peak at No. peak 5.6 ppm difference 6ppm difference 013-1 1 0.9604  −0.00785 0.9435  −0.00335 013-2 1 0.9653 −0.00295 0.9426  −0.00425 013-3 1 0.9726  0.00435 0.9591  0.01225 013-41 0.9973  0.02905 0.9528  0.00595 013-5 1 0.958  −0.01025 0.9478 0.00095 013-6 1 0.9559  −0.01235 0.9353  −0.01155 average = 0.968250.94685

Impurity profiles for the six reactions are shown in Table 19.

TABLE 19 Impurity profiles. retention peak area 013-1 013-1 013-2 013-2013-3 013-3 RRT time (%) (salt) (liquor) (salt) (liquor) (salt) (liquor)IMP 1 0.62 4.35 0.20 0.06 0.18 0.06 0.16 0.05 0.18 IMP 2 0.64 4.44 0.1250.1 1.44 0.10 1.49 0.03 0.92 IMP 3 0.69 4.81 0.27 0.6 1.44 0.06 1.480.04 1.42 IMP 4 0.91 6.34 1.30 1.11 2.73 1.04 2.88 1.04 2.82 HETrE 16.97 95.67 96.43 85.66 96.67 86.41 96.83 86.73 IMP 5 1.03 7.2 0 0.140.97 0.14 0.92 0.13 0.93 IMP 6 1.1 7.7 1.44 1.41 1.39 1.40 1.46 1.4 1.49IMP 7 1.75 12.25 0.55 0.25 3.97 ND 3.22 0.16 2.69 retention peak area013-4 013-4 013-5 013-5 013-6 013-6 RRT time (%) (salt) (liquor) (salt)(liquor) (salt) (liquor) IMP 1 0.62 4.35 0.20 0.05 0.28 0.05 0.3 0.090.45 IMP 2 0.64 4.44 0.125 0.09 0.92 0.09 0.98 0.11 1.66 IMP 3 0.69 4.810.27 0.05 1.27 0.05 1.3 0.09 1.65 IMP 4 0.91 6.34 1.30 1.05 2.84 1.022.81 1.00 2.88 HETrE 1 6.97 95.67 96.61 87.31 96.61 86.00 96.47 85.85IMP 5 1.03 7.2 0 0.13 0.89 0.13 0.92 0.14 0.86 IMP 6 1.1 7.7 1.44 1.371.53 1.32 1.32 1.31 1.43 IMP 7 1.75 12.25 0.55 0.18 2.05 0.21 3.04 0.252.72

Example 19: Removal of DGLA Under 15-(S)-HETrE Salt Formation Conditions

To determine if salt formation conditions could simultaneously purgeresidual DGLA from the reaction mixture, amounts of DGLA in varioussolvent systems according to Table 20 were doped into 15-(S)-HETrEbefore addition of methanolic lysine according to Example 16.

TABLE 20 Reaction Solvent Solvent DGLA No. (non-polar) (polar) Ratio(wt. %) 017-1 Ethyl acetate Methanol 85:15 1.25 017-2 Ethyl acetateMethanol 85:15 2.5 017-3 Ethyl acetate Methanol 85:15 5 017-4 Isopropylacetate Methanol 85:15 1.25 017-5 Isopropyl acetate Methanol 85:15 2.5017-6 Isopropyl acetate Methanol 85:15 5

Results of the six reactions are summarized in Table 21.

TABLE 21 Filtration Reaction Cake weight Cake purity Filtrate timeapprox. Filter cake Slurry No. (mg) (%) purity (%) (min) appearancemobility Drainage 017-1 868 96.26 84.49 35 W, P 4 3 017-2 928 96.4281.55 35 W, P 4 3 017-3 880 96.36 85.09 35 W, P 4 3 017-4 957 95.7085.79 30 W, P 4 3 017-5 945 95.67 83.93 30 W, P 4 3 017-6 976 95.9283.05 35 W, P 4 3 Filtration time: time for cake to reach semi drystate, not wet Filter cake appearance: appearance of cake after drying,w = white, y = yellow (colour of cake), H = hard, P = powdery Slurrymobility: 1 = thin, very mobile, 2 = medium thick, mobile, 3 = thick buttransfer mostly prior to rinsing, 4 = thick not easily transferable, 5 =very thick, no flow without fresh diluent

The final ratio of lysine to 15-(S)-HETrE was determined by NMR as shownin Table 22.

TABLE 22 Reaction No. lysine peak peak at 5.6 ppm difference peak at 6ppm difference 017-1 1 0.9469 0.009916667 0.9338 0.009116667 017-2 10.9326 −0.004383333 0.9216 −0.003083333 017-3 1 0.9266 −0.0103833330.9093 −0.015383333 017-4 1 0.9482 0.011216667 0.9366 0.011916667 017-51 0.9407 0.003716667 0.9245 −0.000183333 017-6 1 0.9269 −0.0100833330.9223 −0.002383333 average = 0.936983333 0.924683333

Impurity data for the six reactions are shown in Table 23.

TABLE 23 Impurity data. retention peak area 017-1 017-1 017-2 017-2017-3 017-3 RRT time (%) (salt) (liquor) (salt) (liquor) (salt) (liquor)IMP 1 0.62 4.35 0.20 0.04 0.18 0.05 0.22 0.04 0.18 IMP 2 0.64 4.44 0.120.15 21.4 0.16 2.55 0.13 2.20 IMP 3 0.69 4.81 0.27 0.22 2.07 0.22 2.440.21 2.14 IMP 4 0.91 6.34 1.30 1.07 2.78 1.08 2.89 1.01 2.86 HETrE 16.97 95.67 96.29 84.49 96.42 81.55 96.36 85.09 IMP 5 1.03 7.2 0 0.090.84 0.07 1.07 0.07 0.78 IMP 6 1.1 7.7 1.44 1.49 1.65 1.62 1.63 1.621.65 IMP 7 1.75 12.25 0.55 0.21 2.95 0.17 3.51 0.17 2.32 retention peakarea 017-4 017-4 017-5 017-5 017-6 017-6 RRT time (%) (salt) (liquor)(salt) (liquor) (salt) (liquor) IMP 1 0.62 4.35 0.20 0.06 0.28 0.06 0.370.06 0.34 IMP 2 0.64 4.44 0.125 0.10 3.52 0.31 4.12 0.20 4.17 IMP 3 0.694.81 0.27 0.24 3.39 0.30 3.95 0.30 3.98 IMP 4 0.91 6.34 1.30 1.34 2.921.11 3.21 1.03 3.18 HETrE 1 6.97 95.67 95.70 85.79 95.67 83.93 95.9283.01 IMP 5 1.03 7.2 0 0.08 0.77 0.10 0.95 0.07 0.93 IMP 6 1.1 7.7 1.441.62 1.71 1.76 1.56 1.57 1.58 IMP 7 1.75 12.25 0.55 0.23 0.2 0.06 0.030.17 0.04

The DGLA content of the 15-(S)-HETrE lysine salt was determined by CAD,as shown in Table 24.

TABLE 24 DGLA content of 15-(S)-HETrE lysine salt. DGLA in salt ReactionSpiked DGLA Sample conc. (determined) No. (w/w) (mg/mL) Peak area (w/w)DGLA 0.1625 3449760 017-1 1.25 1.51 244710 0.76 017-2 2.5 1.5 4487681.41 017-3 5 1.47 954206 3.06 017-4 1.25 1.49 233803 0.74 017-5 2.5 1.63523225 1.51 017-6 5 1.59 1003375 2.97

Example 20: Reslurry Experiments

An experiment was conducted to determine if the ratio of 15-(S)-HETrE tolysine would change after exposure to ethyl acetate. 15-(S)-HETrE lysinesalt (100 mg) was suspended in HPLC grade ethyl acetate (1 mL) andstirred vigorously overnight at room temperature. The suspension wasthen filtered as described in previous examples. The impurity profilesof the starting 15-(S)-HETrE lysine salt (“before slurry”) and theslurried, filtered salt (“after reslurry”) are shown in Table 25.

TABLE 25 Retention Before After RRT time reslurry reslurry IMP 1 0.624.35 0.04 0.16 IMP 2 0.64 4.44 0.09 0.39 IMP 3 0.69 4.81 0.11 0.16 IMP 40.91 6.34 1.01 1.08 HETrE 1 6.97 96.66 95.69 IMP 5 1.03 7.2 0.15 0.07IMP 6 1.1 7.7 1.39 1.47 IMP 7 1.75 12.25 0.26 0.11

NMR spectroscopy confirmed no change in the ratio of15-(S)-HETrE-to-lysine ratio.

Example 21: Solvent-Induced Degradation of 15-(S)-HETrE Lysine Salt

An experiment to determine the degradation of 15-(S)-HETrE lysine salt,if any, when contacted with ethyl acetate or methanol. 15-(S)-HETrElysine salt (100 mg) was dissolved in solvent (1 mL) and stirredovernight. UPLC analysis of the resulting mixtures, compared to theoriginal 15-(S)-HETrE lysine salt, are shown in Table 26.

TABLE 26 Retention Peak area In In ethyl RRT time (%) methanol acetateIMP 1 0.62 4.35 0.20 0.03 0.05 IMP 2 0.64 4.44 0.12 0.24 0.86 IMP 3 0.694.81 0.27 0.45 0.96 IMP 4 0.91 6.34 1.30 1.16 1.30 HETrE 1 6.97 95.6795.40 94.20 IMP 5 1.03 7.2 0 0.13 0.29 IMP 6 1.1 7.7 1.44 1.34 1.35 IMP7 1.75 12.25 0.55 0.31 0.14

Example 22: Seeding Timing

Experiments to determine timing for efficient addition of seed wereperformed. Two experiments were set up similar to Example 16.15-(S)-HETrE dissolved in ethyl acetate was added as a percentage of thetotal ethyl acetate charge (see Table 27), and was added to a stirringsuspension of lysine hydrate (399±2 mg) in methanol (1.2 mL) andobserved for a period of time.

TABLE 27 15-(S)-HETrE Experiment (% charge) 1 10 2 20

In each of Experiments 1 and 2, addition of 15-(S)-HETrE solution tolysine suspension created a viscous non-mobile suspension.

Example 23: Preparation of 15-(S)-HETrE Methyl Ester

15-(S)-HETrE methyl ester was prepared by combining 15-(S)-HETrE (100mg), potassium carbonate (129 mg, 3 equiv.) and methyl iodide (220 mg, 5equiv.) in DMF (0.5 mL). The mixture was stirred at room temperature for18 hours. Following a quench with water (5 mL), the organic phase wasextracted with MtBE (2×5 mL) and the combined organic phases were washedwith brine (5 mL), dried and concentrated.

Example 24: Preparation of 15-(S)-HETrE Lysine Salt

A series of experiments was performed to determine various processparameters.

Reaction 021-A: L-Lysine Suspended in Methanol

L-Lysine mono-hydrate (798±2 mg) was suspended in degassed methanol (3.2mL) in a nitrogen filled 50 mL 3-neck round bottom flask (RBF).15-(S)-HETrE (1606±2 mg) was dissolved in degassed ethyl acetate (12.8mL) and added to the stirring suspension of lysine. Seed (100 mg) wasadded subsequently and stirred at 250 rpm overnight. The suspension wasfiltered under nitrogen through a size 3 sinter glass funnel (40 mmdiameter) and washed with ethyl acetate (12 mL, 3×4 mL) portions.

Reaction 021-B: L-Lysine Suspended in Ethyl Acetate

L-Lysine mono-hydrate (798±2 mg) was suspended in degassed ethyl acetate(12.8 mL) in a nitrogen filled 50 mL 3-neck RBF. 15-(S)-HETrE (1606±2mg) was dissolved in degassed methanol (3.2 mL) and added to thestirring suspension of lysine. Seed (100 mg) was added subsequently andstirred at 250 rpm overnight. The suspension was filtered under nitrogenthrough a size 3 sinter glass funnel (40 mm diameter) and washed withethyl acetate (12 mL, 3×4 mL) portions.

Reaction 021-C: Addition of Hot 15-(S)-HETrE

An experiment was performed as above, except the 15-(S)-HETrE solutionwas heated to 45° C. before addition to the lysine suspension.

Reaction 021-D: Addition of 15-(S)-HETrE to Hot Lysine Suspension

An experiment was performed as above, except the lysine suspension washeated to 45° C. for 15 minutes to equilibrate before the solution of15-(S)-HETrE was added. The seed (100 mg) was then added, and themixture was stirred for 5 minutes and left cool to room temperature inthe oil bath.

Reaction 021-E: Absence of Seed Addition

An experiment identical to Reaction 021-D was performed except the seedwas not added.

Reaction 021-F: Additional Temperature Cycling

Reaction 021-E was repeated with an addition temperature cycle asfollows: after the salt formation occurred at 45° C., the mixture wascooled to 20° C., then re-heated to 45° C. and held for 2 hours. Finalcool-down to 20° C. occurred overnight.

Reaction 021-G: Additional Stir Time

Reaction 021-E was repeated, except the mixture was cooled from 45° C.to 20° C. and stirred for 6 hours before harvesting the 15-(S)-HETrElysine salt.

Results of Reactions 021-A to 021-G are shown in Table 28.

TABLE 28 Filtration Reaction Cake weight Cake purity time approx. Filtercake Slurry No. Ratio (mgs) (%) (mins) appearance mobility Drainage021-A 80:20 1672 95.34 15 W, P 4 3 021-B 80:20 1587 95.24 15 Y, H (W, P4 3 when cake was broken) 021-C 80:20 1509 94.99 15 Y, H (W, P 4 3 whencake was broken) 021-D 80:20 1678 95.06 10-12 W, P 2 (slurry 2 could bepoured from RBF) 021-E 80:20 1627 95.21 10-12 Y, H (W, P 2 2 when cakewas broken) 021-F 80:20 1498 94.20 10-12 Y, H (W, P 2 2 when cake wasbroken) 021-G 80:20 1680 94.94 15 W, P (yellow 2 2 crust)

The ratio of lysine to 15-(S)-HETrE was determined by NMR, as shown inTable 29.

TABLE 29 peak at 5.6 Reaction No. lysine peak ppm difference peak at 6ppm difference 021-A 1 0.9454 0.0157 0.9034 0.00054 021-B 1 0.95810.0284 0.9221 0.01924 021-C 1 0.8764 −0.0533 0.8629 −0.03996 021-D 10.9249 −0.0048 0.9154 0.01254 021-E 1 0.915 −0.0147 0.8928 −0.01006021-F 1 0.92 −0.0097 0.9016 −0.00126 021-G 1 0.9681 0.9681 0.92180.92180 average = 0.9297 0.902857143

The impurity profiles of each reaction is shown in Table 30.

TABLE 30 Impurity profiles. Retention Peak 021-G 021-G RRT time area (%)021-A 021-B 021-C 021-D 021-E 021-F (salt) (liquor) IMP 1 0.62 4.35 0.120.17 0.22 0.24 0.19 0.17 0.27 ND 0.74 IMP 2 0.64 4.44 0.51 0.08 0.120.10 0.08 0.18 0.20 0.24 1.38 IMP 3 0.66 4.64 0.89 0.33 0.44 0.40 0.380.44 0.55 0.52 3.57 IMP 4 0.69 4.81 0.13 0.18 0.22 0.23 0.21 0.18 0.280.26 0.57 IMP 5 0.72 4.99 1.29 0.19 0.24 0.2 0.19 0.31 0.44 0.46 3.94IMP 6 0.85 5.92 0.16 0.08 0.08 0.13 0.09 0.08 0.12 0.08 0.57 IMP 7 0.916.34 1.17 1.06 0.75 0.95 1.08 0.98 0.96 1.01 1.91 IMP 8 0.98 6.84 0.290.09 0.29 0.16 0.11 0.14 0.14 0.89 0.51 HETrE 1 6.97 91.38 95.34 95.2494.99 95.06 95.21 94.20 94.94 69.51 IMP 9 1.03 7.32 0.18 0.15 0.13 0.140.16 0.16 0.15 0.16 0.85 IMP 10 1.1 7.41 0.23 1.41 1.32 1.26 1.39 1.241.30 1.36 1.00 IMP 11 1.75 12.25 0.88 0.18 0.11 0.23 0.25 0.25 0.38 0.316.63

The salt prepared in Reaction 021-D (400 mg) was reslurried in ethylacetate/methanol (80/20, 2 mL) under the following conditions:

-   -   021-D-A: stirred for 6 hours at 20° C.    -   021-D-B: stirred while ramping temperature from 20° C. to 45°        C., then stirred for 2 hours at 45° C. before cooling to 20° C.        (total stirring time: 6 hours).

NMR and impurity data are shown in Tables 31 and 32, respectively.

TABLE 31 Reaction Lysine peak Peak at 5.6 ppm Difference Peak at 6 ppmDifference 021-D-A 1 1.1043 0.03065 03.9818 −0.00765 021-D-B 1 1.043−0.03065 0.9971 0.00765 Average = 1.07365 0.98945

TABLE 32 Peak area Retention (%) RRT time 021-D 021-D-A 021-D-B IMP 10.62 4.35 0.19 0.22 0.22 IMP 2 0.64 4.44 0.08 0.05 0.05 IMP 3 0.66 4.640.38 0.36 0.35 IMP 4 0.69 4.81 0.21 0.23 0.21 IMP 5 0.72 4.99 0.19 0.110.10 IMP 6 0.85 5.92 0.09 0.08 0.08 IMP 7 0.91 6.34 1.08 1.09 1.04 IMP 80.98 6.84 0.11 0.09 0.09 HETrE 1 6.97 95.06 95.50 95.64 IMP 9 1.03 7.320.16 0.16 0.16 IMP 10 1.1 7.41 1.39 1.42 1.37 IMP 11 1.75 12.25 0.250.16 0.18

Reactions 021-D and 021-E were repeated, with filtration of the finalsalt performed using a pressurized filter. Yield and purity of the salt,NMR data, and impurity profiles are shown in Tables 33-35, respectively.

TABLE 33 Filtration Cake weight Cake purity time approx. Filter cakeSlurry experiment (mgs) (%) (mins) appearance mobility Drainage 029-A1795 94.10 15 Y, H 2 2 (white powder when ground) 029-B 1933 93.83 15 Y,H 2 2 (white powder when ground)

TABLE 34 peak at 5.6 peak at 6 experiment lysine peak ppm difference ppmdifference 029-A 1 1.0165 0.00035 0.9869 −0.00070 029-B 1 1.0158−0.00035 0.9883 0.00070 average = 1.01615 0.9876

TABLE 35 Reten- Peak tion area 029-A 029-A 029-B 029-B RRT time (%)(salt) (liquor) (salt) (liquor) IMP 1 0.62 4.35 0.12 0.23 0.93 0.27 0.93IMP 2 0.64 4.44 0.51 0.32 1.37 0.29 1.37 IMP 3 0.66 4.64 0.89 0.67 3.670.74 3.73 IMP 4 0.69 4.81 0.13 0.24 0.99 0.29 0.99 IMP 5 0.72 4.99 1.290.77 3.97 0.33 3.78 IMP 6 0.85 5.92 0.16 0.02 1.01 0.02 0.96 IMP 7 0.916.34 1.17 1.05 1.93 1.06 1.95 IMP 8 0.98 6.84 0.29 0.26 0.65 0.21 0.65HETrE 1 6.97 91.38 94.10 65.65 93.83 66.17 IMP 9 1.03 7.32 0.18 0.190.13 0.18 0.13 IMP 10 1.1 7.41 1.41 1.42 1.26 1.34 1.36 IMP 11 1.7512.25 0.88 0.36 6.99 0.01 6.92

Example 25: Wash Solvents

An experiment was performed to determine the effects of different washsolvents (see Table 36) on the yield and purity of the 15-(S)-HETrElysine salt.

TABLE 36 Filtration Wash Cake weight Cake purity time approx. Filtercake Slurry experiment solvent (mgs) (%) (mins) appearance mobilityDrainage 023-A Ethyl acetate: 111 94.32 10 Y, H (white 2 2 methanolpowder when (80/20) ground) 023-B Ethyl acetate 826 94.10 10 Y, H (white2 2 powder when ground) 023-C methanol 760 94.34 10 Y, H (white 2 2powder when ground)

NMR and impurity data are shown in Tables 37 and 38, respectively.

TABLE 37 peak at 5.6 experiment lysine peak ppm difference peak at 6 ppmdifference 023-A 1 1.0124 0.000933333 0.9814 −0.00707 023-B 1 1.0104−0.001066667 0.9965 0.00803 023-C 1 1.0116 0.000133333 0.9875 −0.00097average = 1.011466667 0.988466667

TABLE 38 Retention Peak area RRT time (%) 023-A 023-B 023-C IMP 1 0.624.35 0.12 0.01 0.01 0.01 IMP 2 0.64 4.44 0.51 0.38 0.38 0.38 IMP 3 0.664.64 0.89 0.66 0.65 0.62 IMP 4 0.69 4.81 0.13 0.40 0.40 0.40 IMP 5 0.724.99 1.29 0.48 0.48 0.51 IMP 6 0.85 5.92 0.16 0.03 0.02 0.04 IMP 7 0.916.34 1.17 1.00 1.07 1.18 IMP 8 0.98 6.84 0.29 0.23 0.21 0.21 HETrE 16.97 91.38 94.32 94.10 94.34 IMP 9 1.03 7.32 0.18 0.18 0.18 0.19 IMP 101.1 7.41 1.41 1.4 1.40 1.49 IMP 11 1.75 12.25 0.88 0.33 0.47 0.52

Example 26: Isopropyl Acetate

An experiment was conducted to examine whether any degradation would beobserved upon exposure of 15-(S)-HETrE to isopropyl acetate.15-(S)-HETrE (100 mg) was dissolved in isopropyl acetate (1 mL) andstirred overnight. This was sampled directly for UPLC analysis (Table39).

TABLE 39 Retention Peak area RRT time (%) Example 26 IMP 1 0.62 4.350.12 0.22 IMP 2 0.64 4.44 0.51 1.48 IMP 3 0.66 4.64 0.89 1.47 IMP 4 0.694.81 0.13 0.19 IMP 5 0.72 4.99 1.29 3.02 IMP 6 0.85 5.92 0.16 0.43 IMP 70.91 6.34 1.17 1.56 IMP 8 0.98 6.84 0.29 0.62 HETrE 1 6.97 91.38 85.56IMP 9 1.03 7.32 0.18 0.28 IMP 10 1.1 7.41 1.41 1.79 IMP 11 1.75 12.250.88 1.14

Example 27: Lysine Salt Formations of Semi-Purified and Crude15-(S)-HETrE

An experiment to determine the impact of salt formation on the purity ofthe 15-(S)-HETrE starting material was performed. The amount of lysineused was adjusted based on the purity of the 15-(S)-HETrE as determinedby UPLC analysis. 15-(S)-HETrE (10 g) was loaded onto a pre-wet columnand eluted with cyclohexane:MtBE (50:50) (350 mL) until the 15-(S)-HETrEspot was no longer evident by TLC. The relevant fractions wereconcentrated. Yield and purity data are shown in Table 40; NMR andimpurity profiles are shown in Tables 41-42, respectively.

TABLE 40 Filtration 15-(S)- Cake weight Cake purity time approx. Filtercake Slurry experiment HETrE (mgs) (%) (mins) appearance mobilityDrainage 027-A Crude 1896 94.75 7 Y, H 2 1 (91.6% (white purity) powderwhen ground) 027-B Semi- 1820 95.37 15 Y, H 2 2 purified (white (92.3%powder when purity) ground)

TABLE 41 peak at 5.6 peak at 6 experiment lysine peak ppm difference ppmdifference 027-A 1 1.0014 −0.0082 0.994 −0.01140 027-B 1 1.0178 0.00821.0168 0.01140 average = 1.0096 1.0054

TABLE 42 Retention Peak area 027-A 027-A Peak area 027-B 027-B RRT time(Crude), % (salt) (liquor) (semipure), % (salt) (liquor) IMP 1 0.62 4.350.05 0.07 0.52 0.09 0.03 0.26 IMP 2 0.64 4.44 0.05 0.05 0.34 0.10 0.050.2 IMP 3 0.66 4.64 0.55 0.57 15.68 0.63 0.24 18.84 IMP 4 0.69 4.81 0.130.17 0.63 0.14 0.12 0.62 IMP 5 0.72 4.99 0.15 0.15 0.84 0.1 0.27 0.14IMP 6 0.85 5.92 0.84 0.14 17.40 0.08 0.09 21.78 IMP 7 0.91 6.34 1.181.09 0.95 1.09 1.14 0.56 IMP 8 0.98 6.84 0.05 0.01 0.98 0.01 0.01 0.02HETrE 1 6.97 91.6 94.75 28.09 92.3 95.37 20.37 IMP 9 1.03 7.32 0.31 0.15.50 0.36 0.19 6.45 IMP 10 1.1 7.41 1.48 1.38 7.21 1.46 1.45 8.58 IMP 111.75 12.25 0.35 0.24 4.88 0.56 0.43 5.68

Example 28: Salt Formation at 0° C.

L-Lysine mono-hydrate (820 mg) was suspended in ethyl acetate (degassed,3.2 mL) in a 25-mL RBF and heated to 45° C. 15-(S)-HETrE (1606±2 mg) wasdissolved in methanol (12.8 mL) and added to the stirring suspension oflysine. The suspension was allowed to cool to room temperature in theoil bath and the flask was then transferred to a water bath and cooledover a period of time to 0° C., after which it was stirred for a further2 hours. The salt was collected by pressure filtration and washed withthe mother liquor. Yield and filtration performance data are shown inTable 43.

TABLE 43 Filtration Cake time weight Cake purity approx. Filter cakeSlurry (mgs) (%) (mins) appearance mobility Drainage 2009 Not 7-10 W,P(hard 2 1 specified and pale yellow when dried further)

Example 29: Salt Formation in Methanol or Ethanol

L-Lysine mono-hydrate (399±2 mg) was suspended in 1.6 mL of eithermethanol (Reaction 031-A) or ethanol (Reaction 031-B) in a 20 mL snapcap vial, 15-(S)-HETrE (803±2 mg) was dissolved in the same alcohol (6.4mL) and added to the stirring suspension of lysine, and stirred at 250rpm overnight. No salt formation was observed in the methanol solventsystem. Seeding with salt from a previous experiment did not induce saltformation. The lysine salt formed in the ethanol system. Yield andpurity data are shown in Table 44; NMR and impurity profiles are shownin Tables 45-46, respectively.

TABLE 44 Filtration Cake weight Cake purity time approx. Filter cakeSlurry experiment Solvent (mgs) (%) (mins) appearance mobility Drainage031-B ethanol 833 93.13 10 Y, H (white 2 2 powder when ground)

TABLE 45 peak at 5.6 peak at 6 experiment lysine peak ppm difference ppmdifference 031-B 1 1.03 — 1.002 —

TABLE 46 Retention Peak area 031-B RRT time (%) 031-B (salt) (liquor)IMP 1 0.62 4.35 0.12 0.31 1.88 IMP 2 0.64 4.44 0.51 0.60 1.62 IMP 3 0.664.64 0.89 0.62 4.32 IMP 4 0.69 4.81 0.13 0.34 1.73 IMP 5 0.72 4.99 1.290.49 3.39 IMP 6 0.85 5.92 0.16 0.01 0.56 IMP 7 0.91 6.34 1.17 1.01 1.76IMP 8 0.98 6.84 0.29 0.02 0.27 HETrE 1 6.97 91.38 93.13 71.78 IMP 9 1.037.32 0.18 0.15 0.59 IMP 10 1.1 7.41 1.41 1.39 1.19 IMP 11 1.75 12.250.88 0.60 3.6

Example 30: Impurity Profiles from Various 15-(S)-HETrE StartingMaterials

15-(S)-HETrE lysine salts were prepared according to a method consistentwith Example 16 from 15-(S)-HETrE starting materials (abbreviated “FA”below) having various purity levels. The impurity profiles of theresulting lysine salts (abbreviated “HLS” below) are shown in Table 47as determined by UPLC.

TABLE 47 Impurity Profiles. Semi- Retention Purified purified Crude timeFA HLS-1 FA HLS-2 FA HLS-3 Notes IMP 1 4.35 0.01 0.04 0.01 0.03 0.050.07 IMP 2 4.44 0.10 0.06 0.09 0.06 0.06 0.05 IMP 3 4.64 0.16 0.09 0.100.11 0.62 0.56 IMP 4 4.81 0.02 0.04 0.63 0.24 0.78 0.18 IMP 5 4.89 0.010.03 0.01 0.02 0.14 0.17 IMP 6 4.99 0.29 0.11 0.13 0.07 0.16 0.11 IMP 75.44 — — 0.02 0.04 0.06 0.01 (1) IMP 8 5.53 — — 0.06 0.03 0.07 0.05 (1)IMP 9 5.92 0.14 0.02 0.79 0.27 0.84 0.14 (2) IMP 10 6.34 0.03 0.03 0.030.03 0.04 0.04 IMP 11 6.46 1.3 1.04 1.14 1.13 1.22 1.09 HETrE 6.9795.37  96.69  92.28 94.27 91.63 94.61 IMP 12 7.32 0.10 0.14 0.17 0.190.20 0.09 IMP 13 7.41 0.10 0.05 0.37 0.15 0.42 0.10 (2) IMP 14 7.78 1.431.42 1.48 1.44 1.49 1.38 IMP 15 7.98 — — 0.34 0.02 0.55 0.02 (1) IMP 168.311 — — 0.04 — 0.04 0.04 (1) IMP 17 8.57 — — 0.1 0.08 0.10 0.07 (1)IMP 18 9.57 — — 0.07 0.07 0.06 0.04 (1) IMP 19 9.67 — — 0.09 0.07 0.090.09 (1) IMP 20 9.77 — — 0.35 0.29 0.35 0.16 (1) IMP 21 9.93 — — 0.090.06 0.09 0.01 (1) IMP 22 10.31 — — 0.24 0.17 0.24 0.19 (1) IMP 23 12.110.56 0.23 0.56 0.43 0.34 0.24 Table 47 Notes: (1) Impurity that ispurged during column chromatography. (2) Impurity that decreased inconcentration upon salt formation.

FURTHER EXAMPLES Further Example 1

A salt of a 15-lipoxygenase product.

Further Example 2

The salt of Further Example 1, wherein the salt is a pharmaceuticallyacceptable salt.

Further Example 3

The salt of Further Example 1 or Further Example 2, wherein the saltcomprises a lysine salt of the 15-lipoxygenase product.

Further Example 4

The salt of any one of Further Examples 1-3, wherein the salt comprisesa sodium salt of the 15-lipoxygenase product.

Further Example 5

The salt of any one of Further Examples 1-4, wherein the salt comprisesan ornithine salt of the 15-lipoxygenase product.

Further Example 6

The salt of any one of Further Examples 1-5, wherein the salt comprisesa piperazine salt of the 15-lipoxygenase product.

Further Example 7

The salt of any one of Further Examples 1-6, wherein the salt comprisesa meglumine salt of the 15-lipoxygenase product.

Further Example 8

The salt of any one of Further Examples 1-7 further comprising the15-lipoxygenase product in free acid form.

Further Example 9

The salt of any one of Further Examples 1-8, wherein the salt isselected from the group consisting of: sodium, lysine, ornithine,piperazine, meglumine, and combinations thereof.

Further Example 10

The salt of any one of Further Examples 1-9, wherein the salt is sodium.

Further Example 11

The salt of any one of Further Examples 1-9, wherein the salt is lysine.

Further Example 12

The salt of any one of Further Examples 1-9, wherein the salt isornithine.

Further Example 13

The salt of any one of Further Examples 1-9, wherein the salt ispiperazine.

Further Example 14

The salt of any one of Further Examples 1-9, wherein the salt ismeglumine.

Further Example 15

The salt of any one of Further Examples 1-14, wherein the15-lipoxygenase product is selected from the group consisting of:13-HODE, 15-HETrE, 15-OHEPA, 15-HETE, and combinations thereof.

Further Example 16

The salt of any one of Further Examples 1-15, wherein the15-lipoxygenase product is 13-HODE.

Further Example 17

The salt of any one of Further Examples 1-15, wherein the15-lipoxygenase product is 15-HETrE.

Further Example 18

The salt of any one of Further Examples 1-15, wherein the15-lipoxygenase product is 15-OHEPA.

Further Example 19

The salt of any one of Further Examples 1-15, wherein the15-lipoxygenase product is 15-HETE.

Further Example 20

13-Hydroperoxyoctadeca-9Z,11E-dienoic acid sodium salt.

Further Example 21

13-Hydroperoxyoctadeca-9Z,11E-dienoic acid lysine salt.

Further Example 22

13-Hydroperoxyoctadeca-9Z,11E-dienoic acid ornithine salt.

Further Example 23

13-Hydroperoxyoctadeca-9Z,11E-dienoic acid piperazine salt.

Further Example 24

13-Hydroperoxyoctadeca-9Z,11E-dienoic acid meglumine salt.

Further Example 25

15-Hydroperoxy-eicosa-8(Z),11(Z),13(E)-trienoic acid sodium salt.

Further Example 26

15-Hydroperoxy-eicosa-8(Z),11(Z),13(E)-trienoic acid lysine salt.

Further Example 27

15-Hydroperoxy-eicosa-8(Z),11(Z),13(E)-trienoic acid ornithine salt.

Further Example 28

15-Hydroperoxy-eicosa-8(Z),11(Z),13(E)-trienoic acid piperazine salt.

Further Example 29

15-Hydroperoxy-eicosa-8(Z),11(Z),13(E)-trienoic acid meglumine salt.

Further Example 30

15-Hydroperoxy-eicosa-5(Z),8(Z),11(Z),13(E),17(Z)-pentaenoic acid sodiumsalt.

Further Example 31

15-Hydroperoxy-eicosa-5(Z),8(Z),11(Z),13(E),17(Z)-pentaenoic acid lysinesalt.

Further Example 32

15-Hydroperoxy-eicosa-5(Z),8(Z),11(Z),13(E),17(Z)-pentaenoic acidornithine salt.

Further Example 33

15-Hydroperoxy-eicosa-5(Z),8(Z),11(Z),13(E),17(Z)-pentaenoic acidpiperazine salt.

Further Example 34

15-Hydroperoxy-eicosa-5(Z),8(Z),11(Z),13(E),17(Z)-pentaenoic acidmeglumine salt.

Further Example 35

15-Hydroperoxy-5,8,11,13-eicosatetraenoic acid sodium salt.

Further Example 36

15-Hydroperoxy-5,8,11,13-eicosatetraenoic acid lysine salt.

Further Example 37

15-Hydroperoxy-5,8,11,13-eicosatetraenoic acid ornithine salt.

Further Example 38

15-Hydroperoxy-5,8,11,13-eicosatetraenoic acid piperazine salt.

Further Example 39

15-Hydroperoxy-5,8,11,13-eicosatetraenoic acid meglumine salt.

Further Example 40

13-Hydroperoxyoctadeca-9Z,11E-dienoic acid sodium salt.

Further Example 41

13-Hydroxyoctadeca-9Z,11E-dienoic acid lysine salt.

Further Example 42

13-Hydroxyoctadeca-9Z,11E-dienoic acid ornithine salt.

Further Example 43

13-Hydroxyoctadeca-9Z,11E-dienoic acid piperazine salt.

Further Example 44

13-Hydroxyoctadeca-9Z,11E-dienoic acid meglumine salt.

Further Example 45

15-Hydroxy-eicosa-8(Z),11(Z),13(E)-trienoic acid sodium salt.

Further Example 46

15-Hydroxy-eicosa-8(Z),11(Z),13(E)-trienoic acid lysine salt.

Further Example 47

15-Hydroxy-eicosa-8(Z),11(Z),13(E)-trienoic acid ornithine salt.

Further Example 48

15-Hydroxy-eicosa-8(Z),11(Z),13(E)-trienoic acid piperazine salt.

Further Example 49

15-Hydroxy-eicosa-8(Z),11(Z),13(E)-trienoic acid meglumine salt.

Further Example 50

15-Hydroxy-eicosa-5(Z),8(Z),11(Z),13(E),17(Z)-pentaenoic acid sodiumsalt.

Further Example 51

15-Hydroxy-eicosa-5(Z),8(Z),11(Z),13(E),17(Z)-pentaenoic acid lysinesalt.

Further Example 52

15-Hydroxy-eicosa-5(Z),8(Z),11(Z),13(E),17(Z)-pentaenoic acid ornithinesalt.

Further Example 53

15-Hydroxy-eicosa-5(Z),8(Z),11(Z),13(E),17(Z)-pentaenoic acid piperazinesalt.

Further Example 54

15-Hydroxy-eicosa-5(Z),8(Z),11(Z),13(E),17(Z)-pentaenoic acid megluminesalt.

Further Example 55

15-Hydroxy-5,8,11,13-eicosatetraenoic acid sodium salt.

Further Example 56

15-Hydroxy-5,8,11,13-eicosatetraenoic acid lysine salt.

Further Example 57

15-Hydroxy-5,8,11,13-eicosatetraenoic acid ornithine salt.

Further Example 58

15-Hydroxy-5,8,11,13-eicosatetraenoic acid piperazine salt.

Further Example 59

15-Hydroxy-5,8,11,13-eicosatetraenoic acid meglumine salt.

Further Example 60

A composition comprising the salt of any one of Further Examples 1-59.

Further Example 61

A pharmaceutical composition comprising a salt form of a 15-lipoxygenaseproduct.

Further Example 62

The pharmaceutical composition of Further Example 61, wherein the saltform of the 15-lipoxygenase product comprises the salt of any one ofFurther Examples 1-59.

Further Example 63

The pharmaceutical composition of Further Example 61 or Further Example62 further comprising an excipient.

Further Example 64

The pharmaceutical composition of any one of Further Examples 61-63,wherein after storage for at least about 4 weeks, the pharmaceuticalcomposition comprises at least about 98%, at least about 99%, or about100% of an initial amount of the salt form of the 15-lipoxygenaseproduct.

Further Example 65

The pharmaceutical composition of any one of Further Examples 61-64,wherein after storage for at least about 10 weeks, the pharmaceuticalcomposition comprises at least about 90%, at least about 91%, at leastabout 92%, at least about 93%, at least about 94%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, at leastabout 99%, or about 100% of an initial amount of the salt form of the15-lipoxygenase product.

Further Example 66

The pharmaceutical composition of any one of Further Examples 61-65,wherein after storage for at least about 24 weeks, the pharmaceuticalcomposition comprises at least about 86%, at least about 87%, at leastabout 88%, at least about 89%, at least about 90%, at least about 91%,at least about 92%, at least about 93%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,at least about 99%, or about 100% of an initial amount of the salt formof the 15-lipoxygenase product.

Further Example 67

The pharmaceutical composition of any one of Further Examples 64-66,wherein the pharmaceutical composition is stored at 2-8° C., at 20° C.,at 25° C., or at 40° C.

Further Example 68

The pharmaceutical composition of any one of Further Examples 64-67,wherein the pharmaceutical composition is stored at 60% RH or at 75% RH.

Further Example 69

The pharmaceutical composition of any one of Further Examples 61-68,wherein the pharmaceutical composition comprises a therapeuticallyeffective amount of the salt of the 15-lipoxygenase product.

Further Example 70

The pharmaceutical composition of Further Example 69, wherein thetherapeutically effective amount of the salt form of the 15-lipoxygenaseproduct is about 0.1 wt. % to about 20 wt. %.

Further Example 71

The pharmaceutical composition of any one of Further Examples 61-70,wherein the pharmaceutical composition is in a form suitable for topicaladministration.

Further Example 72

The pharmaceutical composition of any one of Further Examples 61-71,wherein the salt form of the 15-lipoxygenase product is the solesignificant active ingredient or the sole active ingredient in thepharmaceutical composition.

Further Example 73

The pharmaceutical composition of any one of Further Examples 61-71further comprising an additional active agent.

Further Example 74

The pharmaceutical composition of Further Example 73, wherein the saltform of the 15-lipoxygenase product and the additional active agent areco-formulated as a single dosage unit.

Further Example 75

The pharmaceutical composition of Further Example 73, wherein the saltform of the 15-lipoxygenase product and the additional active agent areformulated as at least two dosage units for coordinated, combined orconcomitant administration.

Further Example 76

A method of treating a disease or disorder in subject in need thereof,the method comprising administering to the subject the pharmaceuticalcomposition of any one of Further Examples 61-75.

Further Example 77

The method of Further Example 76, wherein the disease or disorder isselected from the group consisting of: acne, erythema, infection, fattyliver, neuropathy, and skin inflammation.

Further Example 78

The method of Further Example 76 or Further Example 77, wherein thepharmaceutical composition is administered to the subject in an amountsufficient to provide a therapeutically effective amount of the saltform of the 15-lipoxygenase product.

Further Example 79

The method of Further Example 78, wherein the therapeutically effectiveamount is about 0.001 mg/kg/day to about 100 mg/kg/day.

What is claimed is:
 1. A lysine salt form of15-hydroxy-8(Z),11(Z),13(E)-eicosatrienoic acid (15-HETrE).
 2. Apharmaceutical composition comprising a lysine salt form of 15hydroxy-8(Z),11(Z),13(E)-eicosatrienoic acid (15-HETrE).
 3. Thepharmaceutical composition of claim 2 further comprising an excipient.4. The pharmaceutical composition of claim 2, wherein the pharmaceuticalcomposition is in a form suitable for topical administration.
 5. Thepharmaceutical composition of claim 2, wherein the pharmaceuticalcomposition is suitable for oral administration.